Embryonic Trophectoderm Secretomics Reveals Chemotactic Migration and Intercellular Communication of Endometrial and Circulating MSCs in Embryonic Implantation

Embryonic implantation is a key step in the establishment of pregnancy. In the present work, we have carried out an in-depth proteomic analysis of the secretome (extracellular vesicles and soluble proteins) of two bovine blastocysts embryonic trophectoderm primary cultures (BBT), confirming different epithelial–mesenchymal transition stages in these cells. BBT-secretomes contain early pregnancy-related proteins and angiogenic proteins both as cargo in EVs and the soluble fraction. We have demonstrated the functional transfer of protein-containing secretome between embryonic trophectoderm and maternal MSC in vitro using two BBT primary cultures eight endometrial MSC (eMSC) and five peripheral blood MSC (pbMSC) lines. We observed that eMSC and pbMSC chemotax to both the soluble fraction and EVs of the BBT secretome. In addition, in a complementary direction, we found that the pattern of expression of implantation proteins in BBT-EVs changes depending on: (i) their epithelial–mesenchymal phenotype; (ii) as a result of the uptake of eMSC- or pbMSC-EV previously stimulated or not with embryonic signals (IFN-τ); (iii) because of the stimulation with the endometrial cytokines present in the uterine fluid in the peri-implantation period.     

圆红冬孢酵母中ARE基因的克隆与表达水平分析

克隆了圆红冬孢酵母R. toruloides中的ARE基因并对转录水平进行了初步分析. 结果表明,克隆得到的ARE基因的DNA序列共2624 bp,含6个内含子,开放阅读框2028 bp,编码675个氨基酸. 在限氮培养基中培养的R. toruloides酵母细胞中,ARE基因的转录表达水平随培养时间增加而提高,推测在酵母R. toruloides中ARE基因可能与油脂积累有关.     

Endocytosis at the Crossroad of Polarity and Signaling Regulation: Learning from Drosophila melanogaster and Beyond

Cellular trafficking through the endosomal–lysosomal system is essential for the transport of cargo proteins, receptors and lipids from the plasma membrane inside the cells and across membranous organelles. By acting as sorting stations, vesicle compartments direct the fate of their content for degradation, recycling to the membrane or transport to the trans-Golgi network. To effectively communicate with their neighbors, cells need to regulate their compartmentation and guide their signaling machineries to cortical membranes underlying these contact sites. Endosomal trafficking is indispensable for the polarized distribution of fate determinants, adaptors and junctional proteins. Conversely, endocytic machineries cooperate with polarity and scaffolding components to internalize receptors and target them to discrete membrane domains. Depending on the cell and tissue context, receptor endocytosis can terminate signaling responses but can also activate them within endosomes that act as signaling platforms. Therefore, cell homeostasis and responses to environmental cues rely on the dynamic cooperation of endosomal–lysosomal machineries with polarity and signaling cues. This review aims to address advances and emerging concepts on the cooperative regulation of endocytosis, polarity and signaling, primarily in Drosophila melanogaster and discuss some of the open questions across the different cell and tissue types that have not yet been fully explored.     

Phase Separation and Mechanical Forces in Regulating Asymmetric Cell Division of Neural Stem Cells

Asymmetric cell division (ACD) of neural stem cells and progenitors not only renews the stem cell population but also ensures the normal development of the nervous system, producing various types of neurons with different shapes and functions in the brain. One major mechanism to achieve ACD is the asymmetric localization and uneven segregation of intracellular proteins and organelles into sibling cells. Recent studies have demonstrated that liquid-liquid phase separation (LLPS) provides a potential mechanism for the formation of membrane-less biomolecular condensates that are asymmetrically distributed on limited membrane regions. Moreover, mechanical forces have emerged as pivotal regulators of asymmetric neural stem cell division by generating sibling cell size asymmetry. In this review, we will summarize recent discoveries of ACD mechanisms driven by LLPS and mechanical forces.     

Upregulation of miR145 and miR126 in EVs from Renal Cells Undergoing EMT and Urine of Diabetic Nephropathy Patients

Simple SummaryDiabetic nephropathy is one of the most frequent complications of diabetes, resulting from diffuse damage to different kidney cells. The identification of subjects at risk is mandatory to prevent its development and provide appropriate therapies reducing the unmanageable evolution towards end-stage kidney disease. The aim of this work was to identify urinary-derived extracellular vesicles (EVs) miRNA cargo to be used as biomarker of kidney damage in diabetic patients. The miRNA profile was then correlated with the molecular mechanism associated with the glomerular and tubular damage using a diabetic-like model. In patients, miR145 and miR126 in urinary EVs increased together with albuminuria. MiR145 and miR126 increased in parallel in EVs from renal epithelial cells undergoing transition to a fibrotic mesenchymal phenotype. These data unveiled a role for miR126 and miR145 as the biomarkers of damage progression and proteinuria development in diabetic nephropathy. AbstractDiabetic nephropathy (DN) is a severe kidney-related complication of type 1 and type 2 diabetes and the most frequent cause of end-stage kidney disease. Extracellular vesicles (EVs) present in the urine mainly derive from the cells of the nephron, thus representing an interesting tool mirroring the kidney’s physiological state. In search of the biomarkers of disease progression, we here assessed a panel of urinary EV miRNAs previously related to DN in type 2 diabetic patients stratified based on proteinuria levels. We found that during DN progression, miR145 and miR126 specifically increased in urinary EVs from diabetic patients together with albuminuria. In vitro, miRNA modulation was assessed in a model of TGF-β1-induced glomerular damage within a three-dimensional perfusion system, as well as in a model of tubular damage induced by albumin and glucose overload. Both renal tubular cells and podocytes undergoing epithelial to mesenchymal transition released EVs containing increased miR145 and miR126 levels. At the same time, miR126 levels were reduced in EVs released by glomerular endothelial cells. This work highlights a modulation of miR126 and miR145 during the progression of kidney damage in diabetes as biomarkers of epithelial to mesenchymal transition.     

The Significance of Epithelial-to-Mesenchymal Transition for Circulating Tumor Cells

Epithelial to mesenchymal transition (EMT) is a process involved in embryonic development, but it also plays a role in remote metastasis formation in tumor diseases. During this process cells lose their epithelial features and adopt characteristics of mesenchymal cells. Thereby single tumor cells, which dissolve from the primary tumor, are enabled to invade the blood vessels and travel throughout the body as so called “circulating tumor cells” (CTCs). After leaving the blood stream the reverse process of EMT, the mesenchymal to epithelial transition (MET) helps the cells to seed in different tissues, thereby generating the bud of metastasis formation. As metastasis is the main reason for tumor-associated death, CTCs and the EMT process are in the focus of research in recent years. This review summarizes what was already found out about the molecular mechanisms driving EMT, the consequences of EMT for tumor cell detection, and suitable markers for the detection of CTCs which underwent EMT. The research work done in this field could open new roads towards combating cancer.     

Repeated Irradiation with γ-Ray Induces Cancer Stemness through TGF-β-DLX2 Signaling in the A549 Human Lung Cancer Cell Line

Cancer stem cells (CSCs) play an important role in cancer recurrence and metastasis. It is suggested that the CSC properties in heterogeneous cancer cells can be induced by ionizing radiation (IR). This study investigated the role of DLX2 in the radioresistance and CSC properties induced by IR in NSCLC cancer cells. Here, A549 cells were exposed to fractionated irradiation at a cumulative dose of 52 Gy (4 Gy × 13 times) for a generation of radioresistant cells. After fractionated irradiation, surviving A549 cells exhibited resistance to IR and enhanced expression of various cancer stem cell markers. They also showed upregulation of mesenchymal molecular markers and downregulation of epithelial molecular markers, correlating with an increase in the migration and invasion. Fractionated irradiation triggered the secretion of TGF-β1 and DLX2 expression. Interestingly, the increased DLX2 following fractionated irradiation seemed to induce the expression of the gene for the EGFR-ligand betacellulin via Smad2/3 signaling. To contrast, DLX2 knockdown dramatically decreased the expression of CSC markers, migration, and proliferation. Moreover, A549 cells expressing DLX2 shRNA formed tumors with a significantly smaller volume compared to those expressing control shDNA in a mouse xenograft assay. These results suggest that DLX2 overexpression in surviving NSCLC cancer cells after fractionated IR exposure is involved in the cancer stemness, radioresistance, EMT, tumor survival, and tumorigenic capability.     

Apicobasal Surfaceome Architecture Encodes for Polarized Epithelial Functionality and Depends on Tumor Suppressor PTEN

The loss of apicobasal polarity during the epithelial-to-mesenchymal transition (EMT) is a hallmark of cancer and metastasis. The key feature of this polarity in epithelial cells is the subdivision of the plasma membrane into apical and basolateral domains, with each orchestrating specific intra- and extracellular functions. Epithelial transport and signaling capacities are thought to be determined largely by the quality, quantity, and nanoscale organization of proteins residing in these membrane domains, the apicobasal surfaceomes. Despite its implications for cancer, drug uptake, and infection, our current knowledge of how the polarized surfaceome is organized and maintained is limited. Here, we used chemoproteomic surfaceome scanning to establish proteotype maps of apicobasal surfaceomes and reveal quantitative distributions of, i.e., surface proteases, phosphatases, and tetraspanins as potential key regulators of polarized cell functionality. We show further that the tumor suppressor PTEN regulates polarized surfaceome architecture and uncover a potential role in collective cell migration. Our differential surfaceome analysis provides a molecular framework to elucidate polarized protein networks regulating epithelial functions and PTEN-associated cancer progression.     

EMT Molecular Signatures of Pancreatic Neuroendocrine Neoplasms

Neuroendocrine neoplasms (NENs) are relatively rare neoplasms occurring predominantly in the gastrointestinal tract and pancreas. Their heterogeneity poses challenges for diagnosis and treatment. There is a paucity of markers for characterisation of NEN tumours. For routine diagnosis, immunohistochemistry of the NEN-specific markers CgA and synaptophysin and the proliferation marker Ki-67 are used. These parameters, however, are qualitative and lack the capacity to fully define the tumour phenotype. Molecules of epithelial–mesenchymal transition (EMT) are potential candidates for improved tumour characterisation. Using qRT-PCR, we measured mRNA levels of 27 tumour markers, including 25 EMT-associated markers, in tumour tissue and matched non-tumour tissues for 13 patients with pancreatic NENs. Tissue from patients with three different grades of tumour had distinctly different mRNA profiles. Of the 25 EMT-associated markers analysed, 17 were higher in G3 tissue relative to matched non-tumour tissue, including CD14, CD24, CD31, CD44, CD45, CD56, CK6, CK7, CK13, CK20, NSE, CDX2, CgA, DAXX, PCNA, laminin and Ki-67. The differences in levels of seven EMT-associated markers, Ki-67, DAXX, CD24, CD44, vimentin, laminin and PDX1 plus CgA and NSE (neuroendocrine markers) enabled a distinct molecular signature for each tumour grade to be generated. EMT molecules differentially expressed in three tumour grades have potential for use in tumour stratification and prognostication and as therapeutic targets for treatment of neuroendocrine cancers, following validation with additional samples.     

Isothiocyanates (ITCs) 1-(Isothiocyanatomethyl)-4-phenylbenzene and 1-Isothiocyanato-3,5-bis(trifluoromethyl)benzene—Aldehyde Dehydrogenase (ALDH) Inhibitors,Decreases Cisplatin Tolerance and Migratory Ability of NSCLC

One of the main treatment modalities for non-small-cell lung cancer (NSCLC) is cisplatin-based chemotherapy. However, the acquisition of cisplatin resistance remains a major problem. Existing chemotherapy regimens are often ineffective against cancer cells expressing aldehyde dehydrogenase (ALDH). As such, there is an urgent need for therapies targeting ALDH-positive cancer cells. The present study compares the anticancer properties of 36 structurally diverse isothiocyanates (ITCs) against NSCLC cells with the ALDH inhibitor disulfiram (DSF). Their potential affinity to ALDH isoforms and ABC proteins was assessed using AutoDockTools, allowing for selection of three compounds presenting the strongest affinity to all tested proteins. The selected ITCs had no impact on NSCLC cell viability (at tested concentrations), but significantly decreased the cisplatin tolerance of cisplatin-resistant variant of A549 (A549CisR) and advanced (stage 4) NSCLC cell line H1581. Furthermore, long-term supplementation with ITC 1-(isothiocyanatomethyl)-4-phenylbenzene reverses the EMT phenotype and migratory potential of A549CisR to the level presented by parental A549 cells, increasing E-Cadherin expression, followed by decreased expression of ABCC1 and ALDH3A1. Our data indicates that the ALDH inhibitors DSF and ITCs are potential adjuvants of cisplatin chemotherapy.     

Emerging Roles of Claudins in Human Cancer

Claudins are major integral membrane proteins of tight junctions. Altered expression of several claudin proteins, in particular claudin-1, -3, -4 and -7, has been linked to the development of various cancers. Although their dysregulation in cancer suggests that claudins play a role in tumorigenesis, the exact underlying mechanism remains unclear. The involvement of claudins in tumor progression was suggested by their important role in the migration, invasion and metastasis of cancer cells in a tissue-dependent manner. Recent studies have shown that they play a role in epithelial to mesenchymal transition (EMT), the formation of cancer stem cells or tumor-initiating cells (CSCs/TICs), and chemoresistance, suggesting that claudins are promising targets for the treatment of chemoresistant and recurrent tumors. A recently identified claudin-low breast cancer subtype that is characterized by the enrichment of EMT and stem cell-like features is significantly associated with disease recurrence, underscoring the importance of claudins as predictors of tumor recurrence. The critical role of epigenetic mechanisms in the regulation of claudin expression indicates the possible application of epigenetic therapy to target claudins. A better understanding of the emerging role of claudins in CSC/TICs and chemoresistance may help to develop therapies against recurrent cancers.     

Concurrent Chemoradiotherapy-Driven Cell Plasticity by miR-200 Family Implicates the Therapeutic Response of Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is a common and fatal malignancy with an increasing incidence worldwide. Over the past decade, concurrent chemoradiotherapy (CCRT) with or without surgery is an emerging therapeutic approach for locally advanced ESCC. Unfortunately, many patients exhibit poor response or develop acquired resistance to CCRT. Once resistance occurs, the overall survival rate drops down rapidly and without proper further treatment options, poses a critical clinical challenge for ESCC therapy. Here, we utilized lab-created CCRT-resistant cells as a preclinical study model to investigate the association of chemoradioresistantresistance with miRNA-mediated cell plasticity alteration, and to determine whether reversing EMT status can re-sensitize refractory cancer cells to CCRT response. During the CCRT treatment course, refractory cancer cells adopted the conversion of epithelial to mesenchymal phenotype; additionally, miR-200 family members were found significantly down-regulated in CCRT resistance cells by miRNA microarray screening. Down-regulated miR-200 family in CCRT resistance cells suppressed E-cadherin expression through snail and slug, and accompany with an increase in N-cadherin. Rescuing expressions of miR-200 family members in CCRT resistance cells, particularly in miR-200b and miR-200c, could convert cells to epithelial phenotype by increasing E-cadherin expression and sensitize cells to CCRT treatment. Conversely, the suppression of miR-200b and miR-200c in ESCC cells attenuated E-cadherin, and that converted cells to mesenchymal type by elevating N-cadherin expression, and impaired cell sensitivity to CCRT treatment. Moreover, the results of ESCC specimens staining established the clinical relevance that higher N-cadherin expression levels associate with the poor CCRT response outcome in ESCC patients. Conclusively, miR-200b and miR-200c can modulate the conversion of epithelial–mesenchymal phenotype in ESCC, and thereby altering the response of cells to CCRT treatment. Targeting epithelial–mesenchymal conversion in acquired CCRT resistance may be a potential therapeutic option for ESCC patients.