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BACKGROUND AIMS microRNAs (miRNAs) are small non-coding RNAs that bind to 3'UTR regions of mRNAs to promote their degradation or block their translation. Mice with disruption of the trefoil factor 1 gene (Tff1) develop gastric neoplasms. We studied these mice to identify conserved miRNA networks involved in gastric carcinogenesis. METHODS We performed next-generation miRNA sequencing analysis of normal gastric tissues (based on histology) from subjects without evidence of gastric neoplasm from patients (n=64) and TFF1-knockout mice (n=22). We validated our findings using 270 normal gastric tissues (including 61 samples from patients without evidence of neoplastic lesions) and 234 gastric tumor tissues from 3 separate cohorts of patients and from mice. We performed molecular and functional assays using cell lines (MKN28,MKN45,STKM2,and AGS cells),gastric organoids,and mice with xenograft tumors. RESULTS We identified 117 miRNAs that were significantly deregulated in mouse and human gastric tumor tissues,compared with non-tumor tissues. We validated changes in levels of 6 miRNAs by quantitative real-time PCR analyses of neoplastic gastric tissues from mice (n=39) and 3 independent cohorts patients (332 patients total). We found levels of MIR135B-5p,MIR196B-5p,and MIR92A-5p to be increased in tumor tissues whereas levels of MIR143-3p,MIR204-5p,and MIR133-3p were decreased in tumor tissues. Levels of MIR143-3p were reduced not only in gastric cancer tissues,but also in normal tissues adjacent to tumors in humans and low-grade dysplasia in mice. Transgenic expression of MIR143-3p in gastric cancer cell lines reduced their proliferation and restored their sensitivity to cisplatin. AGS cells with stable transgenic expression of MIR143-3p grew more slowly as xenograft tumors in mice than control AGS cells; tumor growth from AGS cells that expressed MIR143-3p,but not control cells,was sensitive to cisplatin. We identified and validated bromodomain containing 2 (BRD2) as a direct target of MIR143-3p; increased levels of BRD2 in gastric tumors associated with shorter survival times of patients. CONCLUSIONS In an analysis of miRNA profiles of gastric tumors from mice and human patients,we identified a conserved signature associated with early stages of gastric tumorigenesis. Strategies to restore MIR143-3p or inhibit BRD2 might be developed for treatment of gastric cancer. View Publication

Aneurysms and dissections affecting thoracic aorta are associated with smooth muscle cell (SMC) dysfunction. NO/cGMP signaling pathway in smooth muscle cells has been shown to be affected in sporadic thoracic aortic aneurysms. We analyzed the mRNA levels of PDE5,a cGMP-hydrolyzing enzyme highly expressed in aortic SMCs,that regulates arterious vascular tone by lowering cGMP levels. We found that aortic tissue obtained from Marfan,tricuspid and bicuspid thoracic aneurysms expressed lower levels of PDE5 mRNA compared to control aortas. In particular,we found that affected aortas showed lower levels of all the PDE5A isoforms,compared to control aortas. Transfection of vascular SMCs (VSMCs) with NOTCH3 activated domain (NICD3) induced the expression of PDE5A1 and A3 protein isoforms,but not that of the corresponding mRNAs. VSMC stimulation with GSNO,a nitric oxide analogue or with 8-br-cGMP,but not with 8-br-cAMP,up-regulated PDE5 and NOTCH-3 protein levels,indicating a negative feedback loop to protect the arterial wall from excessive relaxation. Finally,we found that PDE5 is expressed early during human aorta development,suggesting that if loss of function mutations of PDE5 occur,they might potentially affect aortic wall development. View Publication

Multiple myeloma (MM) is a tumor of plasma cells (PCs). Due to the intense immunoglobulin secretion,PCs are prone to endoplasmic reticulum stress and activate several stress-managing pathways,including autophagy. Indeed,autophagy deregulation is maladaptive for MM cells,resulting in cell death. CK1alpha,a pro-survival kinase in MM,has recently been involved as a regulator of the autophagic flux and of the transcriptional competence of the autophagy-related transcription factor FOXO3a in several cancers. In this study,we investigated the role of CK1alpha in autophagy in MM. To study the autophagic flux we generated clones of MM cell lines expressing the mCherry-eGFP-LC3B fusion protein. We observed that CK1 inhibition with the chemical ATP-competitive CK1 alpha/delta inhibitor D4476 resulted in an impaired autophagic flux,likely due to an alteration of lysosomes acidification. However,D4476 caused the accumulation of the transcription factor FOXO3a in the nucleus,and this was paralleled by the upregulation of mRNA coding for autophagic genes. Surprisingly,silencing of CK1alpha by RNA interference triggered the autophagic flux. However,FOXO3a did not shuttle into the nucleus and the transcription of autophagy-related FOXO3a-dependent genes was not observed. Thus,while the chemical inhibition with the dual CK1alpha/delta inhibitor D4476 induced cell death as a consequence of an accumulation of ineffective autophagic vesicles,on the opposite,CK1alpha silencing,although it also determined apoptosis,triggered a full activation of the early autophagic flux,which was then not supported by the upregulation of autophagic genes. Taken together,our results indicate that the family of CK1 kinases may profoundly influence MM cells survival also through the modulation of the autophagic pathway. View Publication

A renewable source of human monocytes and macrophages would be a valuable alternative to primary cells from peripheral blood (PB) in biomedical research. We developed an efficient protocol to derive monocytes and macrophages from human induced pluripotent stem cells (hiPSCs) and performed a functional comparison with PB-derived cells. hiPSC-derived monocytes were functional after cryopreservation and exhibited gene expression profiles comparable with PB-derived monocytes. Notably,hiPSC-derived monocytes were more activated with greater adhesion to endothelial cells under physiological flow. hiPSC-derived monocytes were successfully polarized to M1 and M2 macrophage subtypes,which showed similar pan- and subtype-specific gene and surface protein expression and cytokine secretion to PB-derived macrophages. hiPSC-derived macrophages exhibited higher endocytosis and efferocytosis and similar bacterial and tumor cell phagocytosis to PB-derived macrophages. In summary,we developed a robust protocol to generate hiPSC monocytes and macrophages from independent hiPSC lines that showed aspects of functional maturity comparable with those from PB. View Publication

A proposed strategy to cure HIV uses latency-reversing agents (LRAs) to reactivate latent proviruses for purging HIV reservoirs. A variety of LRAs have been identified,but none has yet proven effective in reducing the reservoir size in vivo. Nanocarriers could address some major challenges by improving drug solubility and safety,providing sustained drug release,and simultaneously delivering multiple drugs to target tissues and cells. Here,we formulated hybrid nanocarriers that incorporate physicochemically diverse LRAs and target lymphatic CD4+ T cells. We identified one LRA combination that displayed synergistic latency reversal and low cytotoxicity in a cell model of HIV and in CD4+ T cells from virologically suppressed patients. Furthermore,our targeted nanocarriers selectively activated CD4+ T cells in nonhuman primate peripheral blood mononuclear cells as well as in murine lymph nodes,and substantially reduced local toxicity. This nanocarrier platform may enable new solutions for delivering anti-HIV agents for an HIV cure. View Publication

Niches in the bone marrow regulate hematopoietic stem and progenitor cell (HSPC) fate and behavior through cell-cell interactions and soluble factor secretion. The niche-HSPC crosstalk is a very complex process not completely elucidated yet. To aid further investigation of this crosstalk,a functional in vitro 3D model that closely represents the main supportive compartments of the bone marrow is developed. Different combinations of human stromal cells and hydrogels are tested for their potential to maintain CD34+ HSPCs. Cell viability,clonogenic hematopoietic potential,and surface marker expression are assessed over time. Optimal HSPC support is obtained in presence of adipogenic and osteogenic cells,together with progenitor derived endothelial cells. When cultured in a bioactive hydrogel,the supportive cells self-assemble into a hypoxic stromal network,stimulating CD34+ CD38+ cell formation,while maintaining the pool of CD34+ 38- HSPCs. HSPC clusters colocalize with the stromal networks,in close proximity to sinusoidal clusters of CD31+ endothelial cells. Importantly,the primary in vitro niche model supports HSPCs with no cytokine addition. Overall,the engineered primary 3D bone marrow environment provides an easy and reliable model to further investigate interactions between HSPCs and their endosteal and perivascular niches,in the context of normal hematopoiesis or blood-related diseases. View Publication

Pathobiology of several chronic inflammatory disorders,including ulcerative colitis and Crohn's disease is related to intermittent,spontaneous injury/ulceration of mucosal surfaces. Disease morbidity has been associated with pathologic release of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha). In this report,we show that TNFalpha promotes intestinal mucosal repair through upregulation of the GPCR platelet activating factor receptor (PAFR) in the intestinal epithelium. Platelet activating factor (PAF) was increased in healing mucosal wounds and its engagement with epithelial PAFR leads to activation of epidermal growth factor receptor,Src and Rac1 signaling to promote wound closure. Consistent with these findings,delayed colonic mucosal repair was observed after administration of a neutralizing TNFalpha antibody and in mice lacking PAFR. These findings suggest that in the injured mucosa,the pro-inflammatory milieu containing TNFalpha and PAF sets the stage for reparative events mediated by PAFR signaling. View Publication

Identifying targets present in the tumor microenvironment that contribute to immune evasion has become an important area of research. In this study,we identified EphB4-ephrin-B2 signaling as a regulator of both innate and adaptive components of the immune system. EphB4 belongs to receptor tyrosine kinase family that interacts with ephrin-B2 ligand at sites of cell-cell contact,resulting in bidirectional signaling. We found that EphB4-ephrin-B2 inhibition alone or in combination with radiation (RT) reduced intratumoral regulatory T cells (Tregs) and increased activation of both CD8+ and CD4+Foxp3- T cells compared with the control group in an orthotopic head and neck squamous cell carcinoma (HNSCC) model. We also compared the effect of EphB4-ephrin-B2 inhibition combined with RT with combined anti-PDL1 and RT and observed similar tumor growth suppression,particularly at early time-points. A patient-derived xenograft model showed reduction of tumor-associated M2 macrophages and favored polarization towards an antitumoral M1 phenotype following EphB4-ephrin-B2 inhibition with RT. In vitro,EphB4 signaling inhibition decreased Ki67-expressing Tregs and Treg activation compared with the control group. Overall,our study is the first to implicate the role of EphB4-ephrin-B2 in tumor immune response. Moreover,our findings suggest that EphB4-ephrin-B2 inhibition combined with RT represents a potential alternative for patients with HNSCC and could be particularly beneficial for patients who are ineligible to receive or cannot tolerate anti-PDL1 therapy. SIGNIFICANCE: These findings present EphB4-ephrin-B2 inhibition as an alternative to anti-PDL1 therapeutics that can be used in combination with radiation to induce an effective antitumor immune response in patients with HNSCC. View Publication

Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate lipid biosynthesis and adipogenesis by controlling the expression of several enzymes required for cholesterol,fatty acid,triacylglycerol and phospholipid synthesis. In vertebrates,SREBP activation is mainly controlled by a complex and well-characterized feedback mechanism mediated by cholesterol,a crucial bio-product of the SREBP-activated mevalonate pathway. In this work,we identified acto-myosin contractility and mechanical forces imposed by the extracellular matrix (ECM) as SREBP1 regulators. SREBP1 control by mechanical cues depends on geranylgeranyl pyrophosphate,another key bio-product of the mevalonate pathway,and impacts on stem cell fate in mouse and on fat storage in Drosophila. Mechanistically,we show that activation of AMP-activated protein kinase (AMPK) by ECM stiffening and geranylgeranylated RhoA-dependent acto-myosin contraction inhibits SREBP1 activation. Our results unveil an unpredicted and evolutionary conserved role of SREBP1 in rewiring cell metabolism in response to mechanical cues. View Publication

The importance of early cancer diagnosis and improved cancer therapy has been clear for years and has initiated worldwide research towards new possibilities in the care strategy of patients with cancer using technological innovations. One of the key research fields involves the separation and detection of circulating tumor cells (CTC) because of their suggested important role in early cancer diagnosis and prognosis,namely,providing easy access by a liquid biopsy from blood to identify metastatic cells before clinically detectable metastasis occurs and to study the molecular and genetic profile of these metastatic cells. Provided the opportunity to further progress the development of technology for treating cancer,several CTC technologies have been proposed in recent years by various research groups and companies. Despite their potential role in cancer healthcare,CTC methods are currently mainly used for research purposes,and only a few methods have been accepted for clinical application because of the difficulties caused by CTC heterogeneity,CTC separation from the blood,and a lack of thorough clinical validation. Therefore,the standardization and clinical application of various developed CTC technologies remain important subsequent necessary steps. Because of their suggested future clinical benefits,we focus on describing technologies using whole blood samples without any pretreatment and discuss their advantages,use,and significance. Technologies using whole blood samples utilize size-based,immunoaffinity-based,and density-based methods or combinations of these methods as well as positive and negative enrichment during separation. Although current CTC technologies have not been truly implemented yet,they possess high potential as future clinical diagnostic techniques for the individualized therapy of patients with cancer. Thus,a detailed discussion of the clinical suitability of these new advanced technologies could help prepare clinicians for the future and can be a foundation for technologies that would be used to eliminate CTCs in vivo. View Publication