Novel Human Tenascin-C Function-Blocking Camel Single Domain Nanobodies.

The extracellular matrix (ECM) molecule Tenascin-C (TNC) is well-known to promote tumor progression by multiple mechanisms. However, reliable TNC detection in tissues of tumor banks remains limited. Therefore, we generated dromedary single-domain nanobodies Nb3 and Nb4 highly specific for human TNC (hTNC) and characterized the interaction with TNC by several approaches including ELISA, western blot, isothermal fluorescence titration and negative electron microscopic imaging. Our results revealed binding of both nanobodies to distinct sequences within fibronectin type III repeats of hTNC. By immunofluroescence and immunohistochemical imaging we observed that both nanobodies detected TNC expression in PFA and paraffin embedded human tissue from ulcerative colitis, solid tumors and liver metastasis. As TNC impairs cell adhesion to fibronectin we determined whether the nanobodies abolished this TNC function. Indeed, Nb3 and Nb4 restored adhesion of tumor and mesangial cells on a fibronectin/TNC substratum. We recently showed that TNC orchestrates the immune-suppressive tumor microenvironment involving chemoretention, causing tethering of CD11c+ myeloid/dendritic cells in the stroma. Here, we document that immobilization of DC2.4 dendritic cells by a CCL21 adsorbed TNC substratum was blocked by both nanobodies. Altogether, our novel TNC specific nanobodies could offer valuable tools for detection of TNC in the clinical practice and may be useful to inhibit the immune-suppressive and other functions of TNC in cancer and other diseases.

Tenascin-W Is a Novel Stromal Marker in Biliary Tract Cancers.

Extrahepatic cancers of the biliary system are typically asymptomatic until after metastasis, which contributes to their poor prognosis. Here we examined intrahepatic cholangiocarcinomas (n = 8), carcinomas of perihilar bile ducts (n = 7), carcinomas of the gallbladder (n = 11) and hepatic metastasis from carcinomas of the gallbladder (n = 4) for the expression of the extracellular matrix glycoproteins tenascin-C and tenascin-W. Anti-tenascin-C and anti-tenascin-W immunoreactivity was found in all biliary tract tumors examined. Unlike tenascin-C, tenascin-W was not detected in normal hepatobiliary tissue. Tenascin-W was also expressed by the cholangiocarcinoma-derived cell line Huh-28. However, co-culture of Huh-28 cells with immortalized bone marrow-derived stromal cells was necessary for the formation and organization of tenascin-W fibrils in vitro. Our results indicate that tenascin-W may be a novel marker of hepatobiliary tumor stroma, and its absence from many normal tissues suggests that it may be a potential target for biotherapies.

Medullary Respiratory Circuit Is Reorganized by a Seasonally-Induced Program in Preparation for Hibernation.

Deep hibernators go through several cycles of profound drops in body temperature during the winter season, with core temperatures sometimes reaching near freezing. Yet unlike non-hibernating mammals, they can sustain breathing rhythms. The physiological processes that make this possible are still not understood. In this study, we focused on the medullary Ventral Respiratory Column of a facultative hibernator, the Syrian hamster. Using shortened day-lengths, we induced a "winter-adapted" physiological state, which is a prerequisite for hibernation. When recording electrophysiological signals from acute slices in the winter-adapted pre-Bötzinger complex (preBötC), spike trains showed higher spike rates, amplitudes, complexity, as well as higher temperature sensitivity, suggesting an increase in connectivity and/or synaptic strength during the winter season. We further examined action potential waveforms and found that the depolarization integral, as measured by the area under the curve, is selectively enhanced in winter-adapted animals. This suggests that a shift in the ion handling kinetics is also being induced by the winter-adaptation program. RNA sequencing of respiratory pre-motor neurons, followed by gene set enrichment analysis, revealed differential regulation and splicing in structural, synaptic, and ion handling genes. Splice junction analysis suggested that differential exon usage is occurring in a select subset of ion handling subunits (ATP1A3, KCNC3, SCN1B), and synaptic structure genes (SNCB, SNCG, RAB3A). Our findings show that the hamster respiratory center undergoes a seasonally-cued alteration in electrophysiological properties, likely protecting against respiratory failure at low temperatures.

Mechanically Defined Microenvironment Promotes Stabilization of Microvasculature, Which Correlates with the Enrichment of a Novel Piezo-1+ Population of Circulating CD11b+ /CD115+ Monocytes.

Vascularization is a critical step in the restoration of cellular homeostasis. Several strategies including localized growth factor delivery, endothelial progenitor cells, genetically engineered cells, gene therapy, and prevascularized implants have been explored to promote revascularization. But, long-term stabilization of newly induced vessels remains a challenge. It has been shown that fibroblasts and mesenchymal stem cells can stabilize newly induced vessels. However, whether an injected biomaterial alone can serve as an instructive environment for angiogenesis remains to be elucidated. It is reported here that appropriate vascular branching, and long-term stabilization can be promoted simply by implanting a hydrogel with stiffness matching that of fibrin clot. A unique subpopulation of circulating CD11b+ myeloid and CD11b+ /CD115+ monocytes that express the stretch activated cation channel Piezo-1, which is enriched prominently in the clot-like hydrogel, is identified. These findings offer evidence for a mechanobiology paradigm in angiogenesis involving an interplay between mechanosensitive circulating cells and mechanics of tissue microenvironment.

mTORC1/autophagy-regulated MerTK in mutant BRAFV600 melanoma with acquired resistance to BRAF inhibition.

BRAF inhibitors (BRAFi) and the combination therapy of BRAF and MEK inhibitors (MEKi) were recently approved for therapy of metastatic melanomas harbouring the oncogenic BRAFV600 mutation. Although these therapies have shown pronounced therapeutic efficacy, the limited durability of the response indicates an acquired drug resistance that still remains mechanistically poorly understood at the molecular level. We conducted transcriptome gene profiling in BRAFi-treated melanoma cells and identified that Mer tyrosine kinase (MerTK) is specifically upregulated. MerTK overexpression was demonstrated not only in melanomas resistant to BRAFi monotherapy (5 out of 10 samples from melanoma patients) but also in melanoma resistant to BRAFi+MEKi (1 out of 3), although MEKi alone does not affect MerTK. Mechanistically, BRAFi-induced activation of Zeb2 stimulates MerTK in BRAFV600 melanoma through mTORC1-triggered activation of autophagy. Co-targeting MerTK and BRAFV600 significantly reduced tumour burden in xenografted mice, which was pheno-copied by co-inhibition of autophagy and mutant BRAFV600.

The expression of tenascin-C and tenascin-W in human ossicles.

The ossicles of the middle ear (the malleus, incus and stapes) transmit forces resulting from vibrations of the tympanic membrane to the cochlea where they are coded as sound. Hearing loss can result from diseases such as rheumatoid arthritis (RA) that affect the joints between the ossicles or degenerative processes like otosclerosis that lead to ankylosis of the footplate of the stapes in the oval window of the cochlea. In this study, immunohistochemistry was used to determine if the extracellular matrix glycoproteins tenascin-C or tenascin-W are expressed in the incudomalleolar and incudostapedial joints of ossicles dissected from human cadavers. Tenascin-C, which is expressed during inflammatory conditions including RA, was seen in the articular cartilage of the incudomalleolar joints and the head of the stapes. Tenascin-W, in contrast, was enriched in the annular ligament that anchors the footplate of the stapes into the oval window of the cochlea.

NDR functions as a physiological YAP1 kinase in the intestinal epithelium.

BACKGROUND: Phosphorylation of the transcriptional coactivator YAP1 is a key event in defining Hippo signaling outputs. Previous studies demonstrated that phosphorylation of YAP1 at serine 127 (S127) sequesters YAP1 in the cytoplasm and consequently inhibits YAP1 transcriptional activity. Mammalian tissue-culture experiments suggest that downstream of MST1/2 signaling, LATS1/2 function as YAP1-S127 kinases. However, studies of Mst1/2 knockout mouse models revealed that the identity of the physiological YAP1-S127 kinase(s) in certain tissues, such as the intestine, remains unknown. RESULTS: We show that mammalian NDR1/2 kinases phosphorylate YAP1 on S127 and thereby negatively regulate YAP1 activity in tissue-cultured cells. By studying NDR1/2-deficient mice, we demonstrate the in vivo relevance of NDR1/2-mediated regulation of YAP1. Specifically, upon loss of NDR1/2 in the intestinal epithelium, endogenous S127 phosphorylation is decreased whereas total YAP1 levels are increased. Significantly, ablation of NDR1/2 from the intestinal epithelium renders mice exquisitely sensitive to chemically induced colon carcinogenesis. Analysis of human colon cancer samples further revealed that NDR2 and YAP1 protein expression are inversely correlated in the majority of samples with high YAP1 expression. Collectively, we report NDR1/2 as physiological YAP1-S127 kinases that might function as tumor suppressors upstream of YAP1 in human colorectal cancer. CONCLUSIONS: We establish mammalian NDR1/2 as bona fide kinases that target YAP1 on S127 in vitro and in vivo. Our findings therefore have important implications for a broad range of research efforts aimed at decoding and eventually manipulating YAP1 biology in cancer settings, regenerative medicine, and possibly also noncancer human diseases.

Tenascin-C is required for normal Wnt/ß-catenin signaling in the whisker follicle stem cell niche.

Whisker follicles have multiple stem cell niches, including epidermal stem cells in the bulge as well as neural crest-derived stem cells and mast cell progenitors in the trabecular region. The neural crest-derived stem cells are a pool of melanocyte precursors. Previously, we found that the extracellular matrix glycoproteins tenascin-C and tenascin-W are expressed near CD34-positive cells in the trabecular stem cell niche of mouse whisker follicles. Here, we analyzed whiskers from tenascin-C knockout mice and found intrafollicular adipocytes and supernumerary mast cells. As Wnt/ß-catenin signaling promotes melanogenesis and suppresses the differentiation of adipocytes and mast cells, we analyzed ß-catenin subcellular localization in the trabecular niche. We found cytoplasmic and nuclear ß-catenin in wild-type mice reflecting active Wnt/ß-catenin signaling, whereas ß-catenin in tenascin-C knockout mice was mostly cell membrane-associated and thus transcriptionally inactive. Furthermore, cells expressing the Wnt/ß-catenin target gene cyclin D1 were enriched in the CD34-positive niches of wild-type compared to tenascin-C knockout mice. We then tested the effects of tenascins on this signaling pathway. We found that tenascin-C and tenascin-W can be co-precipitated with Wnt3a. In vitro, substrate bound tenascins promoted ß-catenin-mediated transcription in the presence of Wnt3a, presumably due to the sequestration and concentration of Wnt3a near the cell surface. We conclude that the presence of tenascin-C in whiskers assures active Wnt/ß-catenin signaling in the niche thereby maintaining the stem cell pool and suppressing aberrant differentiation, while in the knockout mice with reduced Wnt/ß-catenin signaling, stem cells from the trabecular niche can differentiate into ectopic adipocytes and mast cells.