Expression of Epithelial and Mesenchymal Markers in Plasmatic Extracellular Vesicles as a Diagnostic Tool for Neoplastic Processes.

Tumor-derived extracellular vesicles (TD-EVs) have active roles as cancer hallmark enablers. EVs RNA of epithelial and stromal cells carry information that facilitates the communication processes that contribute to oncological progression, so the objective of this work was to validate by RT-PCR the presence of epithelial (KRT19; CEA) and stromal (COL1A2; COL11A1) markers in RNA of plasmatic EVs in healthy and diverse-malignancy patients for the development of a non-invasive cancer diagnosis system using liquid biopsy. Ten asymptomatic controls and 20 cancer patients were included in the study, and results showed that the isolated plasmatic EVs by scanning transmission electron microscopy (STEM) andBiomedical Research Institute A Coruña nanoparticle tracking analysis (NTA) contained most exosome structures with also a considerable percentage of microvesicles. No differences were found in concentration and size distribution between the two cohorts of patients, but significant gene expression in epithelial and mesenchymal markers between healthy donors and patients with active oncological disease was shown. Results of quantitative RT-PCR are solid and reliable for KRT19, COL1A2, and COL11A1, so the analysis of RNA extracted from TD-EVs could be a correct approach to develop a diagnostic tool in oncological processes.

Molecular Detection of Lymph Node Metastases in Lung Cancer Patients Using the One-Step Nucleic Acid Amplification Method:Clinical Significance and Prognostic Value.

The one-step nucleic acid amplification (OSNA) method allows for the quantitative evaluation of the tumor burden in resected lymph nodes (LNs) in patients with lung cancer. This technique enables to detect macro and micrometastases, facilitating the correct classification of patients for appropriate follow-up of the disease after surgery. Of 160 patients with resectable lung cancer whose LNs were examined by OSNA, H&E and CK19 IHC between July 2015 and December 2018, 110 patients with clinical stages from IA1 to IIIB were selected for follow-up. LN staging in lung cancer by pathological study led to understaging in 13.64% of the cases studied. OSNA allowed to quantify the tumor burden and establish a prognostic value. Patients with a total tumor load of ≥1650 cCP/uL were associated with a significantly increased likelihood of recurrence. Moreover, the survival of patients with <4405 cCP/uL was significantly higher than patients with ≥4405 cCP/uL. The OSNA assay is a rapid and accurate technique for quantifying the tumor burden in the LNs of lung cancer patients and OSNA quantitative data could allow to establish prognostic values for recurrence-free survival and overall survival in this type of malignancy.

Molecular Targets in Lung Cancer: Study of the Evolution of Biomarkers Associated with Treatment with Tyrosine Kinase Inhibitors-Has NF1 Tumor Suppressor a Key Role in Acquired Resistance?

The application to clinical practice of liquid biopsy in patients with lung cancer has led to an advance in the diagnosis and monitoring of the disease. Detection of alterations in EGFR genes related to TKI treatment in EGFR-mutated non-small cell lung cancer patients is a routine method in pathology laboratories. The primary objective of this work was to analyze the presence of EGFR mutations in cfDNA of 86 patients with lung cancer undergoing oncological treatment related to response to treatment with TKIs. Secondarily, we evaluated the dynamics of EGFR mutations, the presence of the T790M alteration and its relationship with drug resistance and analyzed by NGS molecular alterations in cfDNA of patients with discordant progression. Our results demonstrate that understanding the mutational status of patients treated with TKIs over time is essential to monitor disease progression. In this context, liquid biopsy is a fundamental key. In addition, it is not only necessary to detect EGFR mutations, but also other concomitant mutations that would be influencing the development of the disease. In this sense, we have discovered that mutations in the NF1 tumor suppressor gene could be exerting an as yet unknown function in lung cancer.

The role of the obestatin/GPR39 system in human gastric adenocarcinomas.

Obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; however, the relationship between the obestatin/GPR39 system and gastric cancer progression remains unknown. In the present study, we determined the expression levels of the obestatin/GPR39 system in human gastric adenocarcinomas and explored their potential functional roles. Twenty-eight patients with gastric adenocarcinomas were retrospectively studied, and clinical data were obtained. The role of obestatin/GPR39 in gastric cancer progression was studied in vitro using the human gastric adenocarcinoma AGS cell line. Obestatin exogenous administration in these GPR39-bearing cells deregulated the expression of several hallmarks of the epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, obestatin signaling promoted phenotypic changes via GPR39, increasingly impacting on the cell morphology, proliferation, migration and invasion of these cells. In healthy human stomachs, obestatin expression was observed in the neuroendocrine cells and GPR39 expression was localized mainly in the chief cells of the oxyntic glands. In human gastric adenocarcinomas, no obestatin expression was found; however, an aberrant pattern of GPR39 expression was discovered, correlating to the dedifferentiation of the tumor. Altogether, our data strongly suggest the involvement of the obestatin/GPR39 system in the pathogenesis and/or clinical outcome of human gastric adenocarcinomas and highlight the potential usefulness of GPR39 as a prognostic marker in gastric cancer.

The NMR structure of human obestatin in membrane-like environments: insights into the structure-bioactivity relationship of obestatin.

The quest for therapeutic applications of obestatin involves, as a first step, the determination of its 3D solution structure and the relationship between this structure and the biological activity of obestatin. On this basis, we have employed a combination of circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, and modeling techniques to determine the solution structure of human obestatin (1). Other analogues, including human non-amidated obestatin (2) and the fragment peptides (6-23)-obestatin (3), (11-23)-obestatin (4), and (16-23)-obestatin (5) have also been scrutinized. These studies have been performed in a micellar environment to mimic the cell membrane (sodium dodecyl sulfate, SDS). Furthermore, structural-activity relationship studies have been performed by assessing the in vitro proliferative capabilities of these peptides in the human retinal pigmented epithelial cell line ARPE-19 (ERK1/2 and Akt phosphorylation, Ki67 expression, and cellular proliferation). Our findings emphasize the importance of both the primary structure (composition and size) and particular segments of the obestatin molecule that posses significant α-helical characteristics. Additionally, details of a species-specific role for obestatin have also been hypothesized by comparing human and mouse obestatins (1 and 6, respectively) at both the structural and bioactivity levels.

The SHP-1 protein tyrosine phosphatase negatively modulates Akt signaling in the ghrelin/GHSR1a system.

The aim of the present study was to identify the signaling mechanism(s) responsible for the modulation of growth hormone secretagogue receptor type 1a (GHSR1a)-associated Akt activity. Ghrelin leads to the activation of Akt through the interplay of distinct signaling mechanisms: an early G(i/o) protein-dependent pathway and a late pathway mediated by ß-arrestins. We found that the Src homology 2-containing protein tyrosine phosphatase (SHP-1) was an essential molecule in both G(i/o) protein-dependent and ß-arrestin-mediated pathways. More specifically, the role of SHP-1 in the G(i/o) protein-dependent pathway was demonstrated by the fact that the overexpression of a catalytically defective SHP-1 augments tyrosine phosphorylation of the PI3K regulatory subunit p85, leading to an increase in the phosphorylation of cSrc and phosphoinositide-dependent protein kinase 1, and finally activating Akt. The presence of SHP-1 in the ß-arrestin-scaffolded complex and its attenuating effect on the cSrc and Akt activities verified that SHP-1 regulates not only the G(i/o) protein-dependent pathway but also the ß-arrestin-mediated pathway. Assays performed in preadipocyte and adipocyte 3T3-L1 cells showed SHP-1 expression. According to our results in HEK-GHSR1a cells, ghrelin stimulated SHP-1 phosphorylation in 3T3-L1 cells. The increase in ghrelin-induced Akt activity was enhanced by small interfering RNA of SHP-1 in preadipocyte 3T3-L1 cells. These results were reproduced in white adipose tissue obtained from mice, in which SHP-1 exhibited higher expression in omental than in subcutaneous tissue. Furthermore, this pattern of expression was inverted in mice fed a high-fat diet, suggesting a role for SHP-1 in controlling ghrelin sensitivity in adipose tissue. Indeed, SHP-1 deficiency was associated with augmented ghrelin-evoked Akt phosphorylation in omental tissue, as well as decreased phosphorylation under overexpression of SHP-1 in subcutaneous tissue. These findings showed a novel role for SHP-1 in the regulation of Akt activity through the modulation of the ghrelin/GHSR1a system signaling.