Blood-derived lncRNAs as biomarkers for cancer diagnosis: the Good, the Bad and the Beauty.

Cancer ranks as one of the deadliest diseases worldwide. The high mortality rate associated with cancer is partially due to the lack of reliable early detection methods and/or inaccurate diagnostic tools such as certain protein biomarkers. Cell-free nucleic acids (cfNA) such as circulating long noncoding RNAs (lncRNAs) have been proposed as a new class of potential biomarkers for cancer diagnosis. The reported correlation between the presence of tumors and abnormal levels of lncRNAs in the blood of cancer patients has notably triggered a worldwide interest among clinicians and oncologists who have been actively investigating their potentials as reliable cancer biomarkers. In this report, we review the progress achieved ("the Good") and challenges encountered ("the Bad") in the development of circulating lncRNAs as potential biomarkers for early cancer diagnosis. We report and discuss the diagnostic performance of more than 50 different circulating lncRNAs and emphasize their numerous potential clinical applications ("the Beauty") including therapeutic targets and agents, on top of diagnostic and prognostic capabilities. This review also summarizes the best methods of investigation and provides useful guidelines for clinicians and scientists who desire conducting their own clinical studies on circulating lncRNAs in cancer patients via RT-qPCR or Next Generation Sequencing (NGS).

Detrimental Effects of IFN-γ on an Epidermolysis Bullosa Simplex Cell Model and Protection by a Humanized Anti-IFN-γ Monoclonal Antibody.

Epidermolysis bullosa is a group of severe skin blistering disorders, which currently have no cure. The pathology of epidermolysis bullosa is recognized as having an inflammatory component, but the role of inflammation in different epidermolysis bullosa disorders is unclear. Epidermolysis bullosa simplex (EBS) is primarily caused by sequence variants in keratin genes; its most severe form, EBS generalized severe, is characterized by aggregates of keratin proteins, and cell models of EBS generalized severe show constitutively elevated stress. IFN-γ is a major mediator of inflammation, and we show that the addition of IFN-γ alone to disease model keratinocytes promotes keratin aggregation, decreases cell-cell junctions, delays wound closure, and reduces cell proliferation. IFN-γ exposure weakens the intercellular cohesion of monolayers on mechanical stress, with IFN-γ-treated EBS monolayers more fragmented than IFN-γ-treated wild-type monolayers. A humanized monoclonal antibody to IFN-γ neutralized the detrimental effects on keratinocytes, restoring cell proliferation, increasing cell-cell adhesion, accelerating wound closure in the presence of IFN-γ, and reducing IFN-γ-mediated keratin aggregation in EBS cells. These suggest that treatment with IFN-γ blocking antibodies may constitute a promising new therapeutic strategy for patients with EBS and may also have ameliorating effects on other inflammatory skin diseases.

A cell-based drug discovery assay identifies inhibition of cell stress responses as a new approach to treatment of epidermolysis bullosa simplex.

In the skin fragility disorder epidermolysis bullosa simplex (EBS), mutations in keratin 14 (K14, also known as KRT14) or keratin 5 (K5, also known as KRT5) lead to keratinocyte rupture and skin blistering. Severe forms of EBS are associated with cytoplasmic protein aggregates, with elevated kinase activation of ERK1 and ERK2 (ERK1/2; also known as MAPK3 and MAPK1, respectively), suggesting intrinsic stress caused by misfolded keratin protein. Human keratinocyte EBS reporter cells stably expressing GFP-tagged EBS-mimetic mutant K14 were used to optimize a semi-automated system to quantify the effects of test compounds on keratin aggregates. Screening of a protein kinase inhibitor library identified several candidates that reduced aggregates and impacted on epidermal growth factor receptor (EGFR) signalling. EGF ligand exposure induced keratin aggregates in EBS reporter keratinocytes, which was reversible by EGFR inhibition. EBS keratinocytes treated with a known EGFR inhibitor, afatinib, were driven out of activation and towards quiescence with minimal cell death. Aggregate reduction was accompanied by denser keratin filament networks with enhanced intercellular cohesion and resilience, which when extrapolated to a whole tissue context would predict reduced epidermal fragility in EBS patients. This assay system provides a powerful tool for discovery and development of new pathway intervention therapeutic avenues for EBS.

Modeling the Structure of Keratin 1 and 10 Terminal Domains and their Misassembly in Keratoderma.

The terminal domains of suprabasal keratins of the skin epithelium are very resistant to evidence-based structural analysis because of their inherent flexibility and lack of predictable structure. We present a model for the structure and interactions of the head and tail domains of epidermal keratins 1 and 10, based on all-atom 3D simulations of keratin primary amino acid sequences, and tyrosine phosphorylation predictions, extracted from published databases. We observed that keratin 1 and 10 end domains are likely to form a tetrameric terminal domain complex incorporating a reversibly extendable region potentially acting as a molecular spring. This structure is formed by intermolecular stacking of aromatic residues, which would spatially constrain the keratin 1/keratin 10 end domains to allow filament compaction and bundling, whilst also retaining extensibility to ensure flexibility of the keratin filament network in the differentiating epidermis. The tetrameric terminal domain complex model may also help to elucidate the effects of mutations in the end domains of suprabasal keratins and so contribute to understanding of the mechanisms leading to keratinopathies such as striate palmoplantar keratoderma, as reported in this study.

TRP channels in brown and white adipogenesis from human progenitors: new therapeutic targets and the caveats associated with the common antibiotic, streptomycin.

Transient receptor potential (TRP) channels are polymodal cell sensors responding to diverse stimuli and widely implicated in the developmental programs of numerous tissues. The evidence for an involvement of TRP family members in adipogenesis, however, is scant. We present the first comprehensive expression profile of all known 27 human TRP genes in mesenchymal progenitors cells during white or brown adipogenesis. Using positive trilineage differentiation as an exclusion criterion, TRP polycystic (P)3, and TPR melastatin (M)8 were found to be uniquely adipospecific. Knockdown of TRPP3 repressed the expression of the brown fat signature genes uncoupling protein (UCP)-1 and peroxisome proliferator-activated receptor γ coactivator (PGC)-1α as well as attenuated forskolin-stimulated uncoupled respiration. However, indices of generalized adipogenesis, such as lipid droplet morphology and fatty acid binding protein (FAPB)-4 expression, were not affected, indicating a principal mitochondrial role of TRPP3. Conversely, activating TRPM8 with menthol up-regulated UCP-1 expression and augmented uncoupled respiration predominantly in white adipocytes (browning), whereas streptomycin antagonized TRPM8-mediated calcium entry, downregulated UCP-1 expression, and mitigated uncoupled respiration; menthol was less capable of augmenting uncoupled respiration (thermogenesis) in brown adipocytes. TRPP3 and TRPM8 hence appear to be involved in the priming of mitochondria to perform uncoupled respiration downstream of adenylate cyclase. Our results also underscore the developmental caveats of using antibiotics in adipogenic studies.-Goralczyk, A., van Vijven, M., Koch, M., Badowski, C., Yassin, M. S., Toh, S.-A., Shabbir, A., Franco-Obregón, A., Raghunath, M. TRP channels in brown and white adipogenesis from human progenitors: new therapeutic targets and the caveats associated with the common antibiotic, streptomycin.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls. The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as "bio-scaffolds" for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications. In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs.

Key to realizing the diagnostic and therapeutic potential of human brown/brite adipocytes is the identification of a renewable, easily accessible and safe tissue source of progenitor cells, and an efficacious in vitro differentiation protocol. We show that macromolecular crowding (MMC) facilitates brown adipocyte differentiation in adult human bone marrow mesenchymal stem cells (bmMSCs), as evidenced by substantially upregulating uncoupling protein 1 (UCP1) and uncoupled respiration. Moreover, MMC also induced 'browning' in bmMSC-derived white adipocytes. Mechanistically, MMC creates a 3D extracellular matrix architecture enshrouding maturing adipocytes in a collagen IV cocoon that is engaged by paxillin-positive focal adhesions also at the apical side of cells, without contact to the stiff support structure. This leads to an enhanced matrix-cell signaling, reflected by increased phosphorylation of ATF2, a key transcription factor in UCP1 regulation. Thus, tuning the dimensionality of the microenvironment in vitro can unlock a strong brown potential dormant in bone marrow.

Assays to Study Consequences of Cytoplasmic Intermediate Filament Mutations: The Case of Epidermal Keratins.

The discovery of the causative link between keratin mutations and a growing number of human diseases opened the way for a better understanding of the function of the whole intermediate filament families of cytoskeleton proteins. This chapter describes analytical approaches to identification and interpretation of the consequences of keratin mutations, from the clinical and diagnostic level to cells in tissue culture. Intermediate filament pathologies can be accurately diagnosed from skin biopsies and DNA samples. The Human Intermediate Filament Database collates reported mutations in intermediate filament genes and their diseases, and can help clinicians to establish accurate diagnoses, leading to disease stratification for genetic counseling, optimal care delivery, and future mutation-aligned new therapies. Looking at the best-studied keratinopathy, epidermolysis bullosa simplex, the generation of cell lines mimicking keratinopathies is described, in which tagged mutant keratins facilitate live-cell imaging to make use of today's powerful enhanced light microscopy modalities. Cell stress assays such as cell spreading and cell migration in scratch wound assays can interrogate the consequences of the compromised cytoskeletal network. Application of extrinsic stresses, such as heat, osmotic, or mechanical stress, can enhance the differentiation of mutant keratin cells from wild-type cells. To bring the experiments to the next level, 3D organotypic human cultures can be generated, and even grafted onto the backs of immunodeficient mice for greater in vivo relevance. While development of these assays has focused on mutant K5/K14 cells, the approaches are often applicable to mutations in other intermediate filaments, reinforcing fundamental commonalities in spite of diverse clinical pathologies.

Making more matrix: enhancing the deposition of dermal-epidermal junction components in vitro and accelerating organotypic skin culture development, using macromolecular crowding.

Skin is one of the most accessible tissues for experimental biomedical sciences, and cultured skin cells represent one of the longest-running clinical applications of stem cell therapy. However, culture-generated skin mimetic multicellular structures are still limited in their application by the time taken to develop these constructs in vitro and by their incomplete differentiation. The development of a functional dermal-epidermal junction (DEJ) is one of the most sought after aspects of cultured skin, and one of the hardest to recreate in vitro. At the DEJ, dermal fibroblasts and epidermal keratinocytes interact to form an interlinked basement membrane of extracellular matrix (ECM), which forms as a concerted action of both keratinocytes and fibroblasts. Successful formation of this basement membrane is essential for take and stability of cultured skin autografts. We studied interactive matrix production by monocultures and cocultures of primary human keratinocytes and fibroblasts in an attempt to improve the efficiency of basement membrane production in culture using mixed macromolecular crowding (mMMC); resulting ECM were enriched with the deposition of collagens I, IV, fibronectin, and laminin 332 (laminin 5) and also in collagen VII, the anchoring fibril component. Our in vitro data point to fibroblasts, rather than keratinocytes, as the major cellular contributors of the DEJ. Not only did we find more collagen VII production and deposition by fibroblasts in comparison to keratinocytes, but also observed that decellularized fibroblast ECM stimulated the production and deposition of collagen VII by keratinocytes, over and above that of keratinocyte monocultures. In confrontation cultures, keratinocytes and fibroblasts showed spontaneous segregation and demarcation of cell boundaries by DEJ protein deposition. Finally, mMMC was used in a classical organotypic coculture protocol with keratinocytes seeded over fibroblast-containing collagen gels. Applied during the submerged phase, mMMC was sufficient to accelerate the emergence of collagen VII along the de novo DEJ, together with stronger transglutaminase activity in the neoepidermis. Our findings corroborate the role of fibroblasts as important players in producing collagen VII and inducing collagen VII deposition in the DEJ, and that macromolecular crowding leads to organotypic epidermal differentiation in tissue culture in a significantly condensed time frame.

ß1A integrin is a master regulator of invadosome organization and function.

Invadosomes are adhesion structures involved in tissue invasion that are characterized by an intense actin polymerization-depolymerization associated with ß1 and ß3 integrins and coupled to extracellular matrix (ECM) degradation activity. We induced the formation of invadosomes by expressing the constitutive active form of Src, SrcYF, in different cell types. Use of ECM surfaces micropatterned at the subcellular scale clearly showed that in mesenchymal cells, integrin signaling controls invadosome activity. Using ß1⁻/⁻ or ß3⁻/⁻ cells, it seemed that ß1A but not ß3 integrins are essential for initiation of invadosome formation. Protein kinase C activity was shown to regulate autoassembly of invadosomes into a ring-like metastructure (rosette), probably by phosphorylation of Ser785 on the ß1A tail. Moreover, our study clearly showed that ß1A links actin dynamics and ECM degradation in invadosomes. Finally, a new strategy based on fusion of the photosensitizer KillerRed to the ß1A cytoplasmic domain allowed specific and immediate loss of function of ß1A, resulting in disorganization and disassembly of invadosomes and formation of focal adhesions.

Podosome-type adhesions and focal adhesions, so alike yet so different.

Cell-matrix adhesions are essential for cell migration, tissue organization and differentiation, therefore playing central roles in embryonic development, remodeling and homeostasis of tissues and organs. Matrix adhesion-dependent signals cooperate with other pathways to regulate biological functions such as cell survival, cell proliferation, wound healing, and tumorigenesis. Cell migration and invasion are integrated processes requiring the continuous, coordinated assembly and disassembly of integrin-mediated adhesions. An understanding of how integrins regulate cell migration and invasiveness through the dynamic regulation of adhesions is fundamental to both physiological and pathological situations. A variety of cell-matrix adhesions has been identified, namely, focal complexes, focal adhesions, fibrillar adhesions, podosomes, and invadopodia (podosome-type adhesions). These adhesion sites contain integrin clusters able to develop specialized structures, which are different in their architecture and dynamics although they share almost the same proteins. Here we compare recent advances and developments in the elucidation of the organization and dynamics of focal adhesions and podosome-type adhesions, in order to understand how such subcellular sites - though closely related in their composition - can be structurally and functionally different. The underlying question is how their respective physiological or pathological roles are related to their distinct organization.

Paxillin phosphorylation controls invadopodia/podosomes spatiotemporal organization.

In Rous sarcoma virus (RSV)-transformed baby hamster kidney (BHK) cells, invadopodia can self-organize into rings and belts, similarly to podosome distribution during osteoclast differentiation. The composition of individual invadopodia is spatiotemporally regulated and depends on invadopodia localization along the ring section: the actin core assembly precedes the recruitment of surrounding integrins and integrin-linked proteins, whereas the loss of the actin core was a prerequisite to invadopodia disassembly. We have shown that invadopodia ring expansion is controlled by paxillin phosphorylations on tyrosine 31 and 118, which allows invadopodia disassembly. In BHK-RSV cells, ectopic expression of the paxillin mutant Y31F-Y118F induces a delay in invadopodia disassembly and impairs their self-organization. A similar mechanism is unraveled in osteoclasts by using paxillin knockdown. Lack of paxillin phosphorylation, calpain or extracellular signal-regulated kinase inhibition, resulted in similar phenotype, suggesting that these proteins belong to the same regulatory pathways. Indeed, we have shown that paxillin phosphorylation promotes Erk activation that in turn activates calpain. Finally, we observed that invadopodia/podosomes ring expansion is required for efficient extracellular matrix degradation both in BHK-RSV cells and primary osteoclasts, and for transmigration through a cell monolayer.