Blood Plasma Circulating DNA-Protein Complexes: Involvement in Carcinogenesis and Prospects for Liquid Biopsy of Breast Cancer.

Circulating DNA (cirDNA) is a promising tool in translational medicine. However, studies of cirDNA have neglected its association with proteins, despite ample evidence that this interaction may affect the fate of DNA in the bloodstream and its molecular functions. The goal of the current study is to shed light on the differences between the proteomic cargos of histone-containing nucleoprotein complexes (NPCs) from healthy female (HFs) and breast cancer patients (BCPs), and to reveal the proteins involved in carcinogenesis. NPCs were isolated from the blood samples of HFs and BCPs using affinity chromatography. A total of 177 and 169 proteins were identified in NPCs from HFs and BCPs using MALDI-TOF mass spectrometry. A bioinformatics analysis revealed that catalytically active proteins, as well as proteins that bind nucleic acids and regulate the activity of receptors, are the most represented among the unique proteins of blood NPCs from HFs and BCPs. In addition, the proportion of proteins participating in ion channels and proteins binding proteins increases in the NPCs from BCP blood. However, the involvement in transport and signal transduction was greater in BCP NPCs compared to those from HFs. Gene ontology term (GO) analysis revealed that the NPC protein cargo from HF blood was enriched with proteins involved in the negative regulation of cell proliferation, and in BCP blood, proteins involved in EMT, invasion, and cell migration were observed. The combination of SPG7, ADRB1, SMCO4, PHF1, and PSMG1 NPC proteins differentiates BCPs from HFs with a sensitivity of 100% and a specificity of 80%. The obtained results indirectly indicate that, in tandem with proteins, blood cirDNA is an important part of intercellular communication, playing a regulatory and integrating role in the physiology of the body.

Comparative Analysis of Molecular Functions and Biological Role of Proteins from Cell-Free DNA-Protein Complexes Circulating in Plasma of Healthy Females and Breast Cancer Patients.

Cell-free DNA (cfDNA) circulates in the bloodstream packed in membrane-coated structures (such as apoptotic bodies) or bound to proteins. To identify proteins involved in the formation of deoxyribonucleoprotein complexes circulating in the blood, native complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies from plasma of healthy females (HFs) and breast cancer patients (BCPs). It was found that the nucleoprotein complexes (NPCs) from HF plasma samples contained shorter DNA fragments (~180 bp) than BCP NPCs. However, the share of DNA in the NPCs from cfDNA in blood plasma in HFs and BCPs did not differ significantly, as well as the share of NPC protein from blood plasma total protein. Proteins were separated by SDS-PAGE and identified by MALDI-TOF mass spectrometry. Bioinformatic analysis showed that in the presence of a malignant tumor, the proportion of proteins involved in ion channels, protein binding, transport, and signal transduction increased in the composition of blood-circulating NPCs. Moreover, 58 (35%) proteins are differentially expressed in a number of malignant neoplasms in the NPCs of BCPs. Identified NPC proteins from BCP blood can be recommended for further testing as breast cancer diagnostic/prognostic biomarkers or as being useful in developing gene-targeted therapy approaches.

Exosomes in Breast Cancer: Involvement in Tumor Dissemination and Prospects for Liquid Biopsy.

In women, breast cancer (BC) is the most commonly diagnosed cancer (24.5%) and the leading cause of cancer death (15.5%). Understanding how this heterogeneous disease develops and the confirm mechanisms behind tumor progression is of utmost importance. Exosomes are long-range message vesicles that mediate communication between cells in physiological conditions but also in pathology, such as breast cancer. In recent years, there has been an exponential rise in the scientific studies reporting the change in morphology and cargo of tumor-derived exosomes. Due to the transfer of biologically active molecules, such as RNA (microRNA, long non-coding RNA, mRNA, etc.) and proteins (transcription factors, enzymes, etc.) into recipient cells, these lipid bilayer 30-150 nm vesicles activate numerous signaling pathways that promote tumor development. In this review, we attempt to shed light on exosomes' involvement in breast cancer pathogenesis (including epithelial-to-mesenchymal transition (EMT), tumor cell proliferation and motility, metastatic processes, angiogenesis stimulation, and immune system repression). Moreover, the potential use of exosomes as promising diagnostic biomarkers for liquid biopsy of breast cancer is also discussed.

Comparative Analysis of Tumor-Associated microRNAs and Tetraspanines from Exosomes of Plasma and Ascitic Fluids of Ovarian Cancer Patients.

Ovarian cancer (OC) is one of the most common and fatal types of gynecological cancer. In the early phase of OC detection, the current treatment and diagnostic methods are not efficient and sensitive enough. Therefore, it is crucial to explore the mechanisms of OC metastasis and discover valuable factors for early diagnosis of female cancers and novel therapeutic strategies for metastasis. Exosomes are known to be involved in the development, migration, and invasion of cancer cells, and their cargo could be useful for the non-invasive biopsy development. CD151- and Tspan8-positive exosomes are known to support the degradation of the extracellular matrix, and are involved in stroma remodeling, angiogenesis and cell motility, as well as the association of miR-24 and miR-101 with these processes. The objective of this study was to explore the relationship of these components of exosomal cargo, in patients with OC, to clarify the clinical significance of these markers in liquid biopsies. The levels of tetraspanins Tspan8+ and CD151+ exosomes were significantly higher in plasma exosomes of OC patients compared with healthy females (HFs). The relative levels of miR-24 and miR-101 in plasma exosomes of HFs were significantly higher than in plasma exosomes of OC patients, while the levels of these microRNAs in exosomes from plasma and ascites of ill females showed no difference. Our study revealed a strong direct correlation between the change in the ascites exosomes CD151+Tspan8+ subpopulation level and the expression levels of the ascites (R = 0.81, p < 0.05) and plasma exosomal miR-24 (R = 0.74, p < 0.05) in OC patients, which confirms the assumption that exosomal cargo act synergistically to increase cellular motility, affecting cellular processes and signaling. Bioinformatics analysis confirmed the involvement of CD151 and Tspan8 tetraspanins and genes controlled by miR-24-3p and miR-101 in signaling pathways, which are crucial for carcinogenesis, demonstrating that these tetraspanins and microRNAs are potential biomarkers for OC screening, and predictors of poor clinicopathological behavior in tumors.