Generation of vestibular tissue-like organoids from human pluripotent stem cells using the rotary cell culture system

Hair cells are specialized mechanosensitive cells responsible for mediating balance and hearing within the inner ear. In mammals, hair cells are limited in number and do not regenerate. Human pluripotent stem cells (hPSCs) provide a valuable source for deriving human hair cells to study their development and design therapies to treat and/or prevent their degeneration. In this study we used a dynamic 3D Rotary Cell Culture System (RCCS) for deriving inner ear organoids from hPSCs. We show RCCS-derived organoids recapitulate stages of inner ear development and give rise to an enriched population of hair cells displaying vestibular-like morphological and physiological phenotypes, which resemble developing human fetal inner ear hair cells as well as the presence of accessory otoconia-like structures. These results show that hPSC-derived organoids can generate complex inner ear structural features and be a resource to study inner ear development

Inhibiting ERK Activation with CI-1040 Leads to Compensatory Upregulation of Alternate MAPKs and Plasminogen Activator Inhibitor-1 following Subtotal Nephrectomy with No Impact on Kidney Fibrosis

Extracellular-signal regulated kinase (ERK) activation by MEK plays a key role in many of the cellular processes that underlie progressive kidney fibrosis including cell proliferation, apoptosis and transforming growth factor β1-mediated epithelial to mesenchymal transition. We therefore assessed the therapeutic impact of ERK1/2 inhibition using a MEK inhibitor in the rat 5/6 subtotal nephrectomy (SNx) model of kidney fibrosis. There was a twentyfold upregulation in phospho-ERK1/2 expression in the kidney after SNx in Male Wistar rats. Rats undergoing SNx became hypertensive, proteinuric and developed progressive kidney failure with reduced creatinine clearance. Treatment with the MEK inhibitor, CI-1040 abolished phospho- ERK1/2 expression in kidney tissue and prevented phospho-ERK1/2 expression in peripheral lymphocytes during the entire course of therapy. CI-1040 had no impact on creatinine clearance, proteinuria, glomerular and tubular fibrosis, and α-smooth muscle actin expression. However, inhibition of ERK1/2 activation led to significant compensatory upregulation of the MAP kinases, p38 and JNK in kidney tissue. CI-1040 also increased the expression of plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasmin-dependent matrix metalloproteinases. Thus inhibition of ERK1/2 activation has no therapeutic effect on kidney fibrosis in SNx possibly due to increased compensatory activation of the p38 and JNK signalling pathways with subsequent upregulation of PAI-1

Propentofylline Targets TROY, a Novel Microglial Signaling Pathway

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain cancer, with a median survival of less than 2 years after diagnosis with current available therapies. The tumor microenvironment serves a critical role in tumor invasion and progression, with microglia as a critical player. Our laboratory has previously demonstrated that propentofylline, an atypical methylxanthine with central nervous system glial modulating and anti-inflammatory actions, significantly decreases tumor growth in a GBM rodent model by preferentially targeting microglia. In the present study, we used the CNS-1 rat glioma model to elucidate the mechanisms of propentofylline. Here we demonstrate that propentofylline targets TROY, a novel signaling molecule up-regulated in infiltrating microglia, and not macrophages, in response to CNS-1 cells. We identify Pyk2, Rac1 and pJNK as the downstream signaling molecules of TROY through western blot analysis and siRNA transfection. We demonstrate that inhibition of TROY expression in microglia by siRNA transfection significantly inhibits microglial migration towards CNS-1 cells similar to 10 µM propentofylline treatment. These results identify TROY as a novel molecule expressed in microglia, involved in their migration and targeted by propentofylline. Furthermore, these results describe a signaling molecule that is differentially expressed between microglia and macrophages in the tumor microenvironment

Modulation of Cell Adhesion and Migration by the Histone Methyltransferase Subunit mDpy-30 and Its Interacting Proteins

We have previously shown that a subset of mDpy-30, an accessory subunit of the nuclear histone H3 lysine 4 methyltransferase (H3K4MT) complex, also localizes at the trans-Golgi network (TGN), where its recruitment is mediated by the TGN-localized ARF guanine nucleotide exchange factor (ArfGEF) BIG1. Depletion of mDpy-30 inhibits the endosome-to-TGN transport of internalized CIMPR receptors and concurrently promotes their accumulation at the cell protrusion. These observations suggest mDpy-30 may play a novel role at the crossroads of endosomal trafficking, nuclear transcription and adhesion/migration. Here we provide novel mechanistic and functional insight into this association. First, we demonstrate a direct interaction between mDpy-30 and BIG1 and locate the binding region in the N-terminus of BIG1. Second, we provide evidence that the depletion or overexpression of mDpy-30 enhances or inhibits cellular adhesion/migration of glioma cells in vitro, respectively. A similar increase in cell adhesion/migration is observed in cells with reduced levels of BIG1 or other H3K4MT subunits. Third, knockdown of mDpy-30, BIG1, or the RbBP5 H3K4MT subunit increases the targeting of β1 integrin to cell protrusions, and suppression of H3K4MT activity by depleting mDpy-30 or RbBP5 leads to increased protein and mRNA levels of β1 integrin. Moreover, stimulation of cell adhesion/migration via mDpy-30 knockdown is abolished after treating cells with a function-blocking antibody to β1 integrin. Taken together, these data indicate that mDpy-30 and its interacting proteins function as a novel class of cellular adhesion/migration modulators partially by affecting the subcellular distribution of endosomal compartments as well as the expression of key adhesion/migration proteins such as β1 integrin

Human U87 Astrocytoma Cell Invasion Induced by Interaction of βig-h3 with Integrin α5β1 Involves Calpain-2

It is known that βig-h3 is involved in the invasive process of many types of tumors, but its mechanism in glioma cells has not been fully clarified. Using immunofluorescent double-staining and confocal imaging analysis, and co-immunoprecipitation assays, we found that βig-h3 co-localized with integrin α5β1 in U87 cells. We sought to elucidate the function of this interaction by performing cell invasion assays and gelatin zymography experiments. We found that siRNA knockdowns of βig-h3 and calpain-2 impaired cell invasion and MMP secretion. Moreover, βig-h3, integrins and calpain-2 are known to be regulated by Ca2+, and they are also involved in tumor cell invasion. Therefore, we further investigated if calpain-2 was relevant to βig-h3-integrin α5β1 interaction to affect U87 cell invasion. Our data showed that βig-h3 co-localized with integrin α5β1 to enhance the invasion of U87 cells, and that calpain-2, is involved in this process, acting as a downstream molecule

Tumour cells expressing single VEGF isoforms display distinct growth, survival and migration characteristics

Vascular endothelial growth factor-A (VEGF) is produced by most cancer cells as multiple isoforms, which display distinct biological activities. VEGF plays an undisputed role in tumour growth, vascularisation and metastasis; nevertheless the functions of individual isoforms in these processes remain poorly understood. We investigated the effects of three main murine isoforms (VEGF188, 164 and 120) on tumour cell behaviour, using a panel of fibrosarcoma cells we developed that express them individually under endogenous promoter control. Fibrosarcomas expressing only VEGF188 (fs188) or wild type controls (fswt) were typically mesenchymal, formed ruffles and displayed strong matrix-binding activity. VEGF164- and VEGF120-producing cells (fs164 and fs120 respectively) were less typically mesenchymal, lacked ruffles but formed abundant cell-cell contacts. On 3D collagen, fs188 cells remained mesenchymal while fs164 and fs120 cells adopted rounded/amoeboid and a mix of rounded and elongated morphologies respectively. Consistent with their mesenchymal characteristics, fs188 cells migrated significantly faster than fs164 or fs120 cells on 2D surfaces while contractility inhibitors accelerated fs164 and fs120 cell migration. VEGF164/VEGF120 expression correlated with faster proliferation rates and lower levels of spontaneous apoptosis than VEGF188 expression. Nevertheless, VEGF188 was associated with constitutively active/phosphorylated AKT, ERK1/2 and Stat3 proteins. Differences in proliferation rates and apoptosis could be explained by defective signalling downstream of pAKT to FOXO and GSK3 in fs188 and fswt cells, which also correlated with p27/p21 cyclin-dependent kinase inhibitor over-expression. All cells expressed tyrosine kinase VEGF receptors, but these were not active/activatable suggesting that inherent differences between the cell lines are governed by endogenous VEGF isoform expression through complex interactions that are independent of tyrosine kinase receptor activation. VEGF isoforms are emerging as potential biomarkers for anti-VEGF therapies. Our results reveal novel roles of individual isoforms associated with cancer growth and metastasis and highlight the importance of understanding their diverse actions

Fibronectin Matrix Assembly Suppresses Dispersal of Glioblastoma Cells

Glioblastoma (GBM), the most aggressive and most common form of primary brain tumor, has a median survival of 12–15 months. Surgical excision, radiation and chemotherapy are rarely curative since tumor cells broadly disperse within the brain. Preventing dispersal could be of therapeutic benefit. Previous studies have reported that increased cell-cell cohesion can markedly reduce invasion by discouraging cell detachment from the tumor mass. We have previously reported that α5β1 integrin-fibronectin interaction is a powerful mediator of indirect cell-cell cohesion and that the process of fibronectin matrix assembly (FNMA) is crucial to establishing strong bonds between cells in 3D tumor-like spheroids. Here, we explore a potential role for FNMA in preventing dispersal of GBM cells from a tumor-like mass. Using a series of GBM-derived cell lines we developed an in vitro assay to measure the dispersal velocity of aggregates on a solid substrate. Despite their similar pathologic grade, aggregates from these lines spread at markedly different rates. Spreading velocity is inversely proportional to capacity for FNMA and restoring FNMA in GBM cells markedly reduces spreading velocity by keeping cells more connected. Blocking FNMA using the 70 KDa fibronectin fragment in FNMA-restored cells rescues spreading velocity, establishing a functional role for FNMA in mediating dispersal. Collectively, the data support a functional causation between restoration of FNMA and decreased dispersal velocity. This is a first demonstration that FNMA can play a suppressive role in GBM dispersal

Receptors for Hyaluronic Acid and Poliovirus: A Combinatorial Role in Glioma Invasion?

Background: CD44 has long been associated with glioma invasion while, more recently, CD155 has been implicated in playing a similar role. Notably, these two receptors have been shown closely positioned on monocytes. Methods and Findings: In this study, an up-regulation of CD44 and CD155 was demonstrated in established and earlypassage cultures of glioblastoma. Total internal reflected fluorescence (TIRF) microscopy revealed close proximity of CD44 and CD155. CD44 antibody blocking and gene silencing (via siRNA) resulted in greater inhibition of invasion than that for CD155. Combined interference resulted in 86 % inhibition of invasion, although in these investigations no obvious evidence of synergy between CD44 and CD155 in curbing invasion was shown. Both siRNA-CD44 and siRNA-CD155 treated cells lacked processes and were rounder, while live cell imaging showed reduced motility rate compared to wild type cells. Adhesion assay demonstrated that wild type cells adhered most efficiently to laminin, whereas siRNA-treated cells (p,0.0001 for both CD44 and CD155 expression) showed decreased adhesion on several ECMs investigated. BrdU assay showed a higher proliferation of siRNA-CD44 and siRNA-CD155 cells, inversely correlated with reduced invasion. Confocal microscopy revealed overlapping of CD155 and integrins (b1, avb1 and avb3) on glioblastoma cell processes whereas siRNAtransfected cells showed consequent reduction in integrin expression with no specific staining patterns. Reduced expression of Rho GTPases, Cdc42, Rac1/2/3, RhoA and RhoB, was seen in siRNA-CD44 and siRNA-CD155 cells. In contrast t

Apc Mutation Enhances PyMT-Induced Mammary Tumorigenesis

The Adenomatous Polyposis Coli (APC) tumor suppressor gene is silenced by hypermethylation or mutated in up to 70% of human breast cancers. In mouse models, Apc mutation disrupts normal mammary development and predisposes to mammary tumor formation; however, the cooperation between APC and other mutations in breast tumorigenesis has not been studied. To test the hypothesis that loss of one copy of APC promotes oncogene-mediated mammary tumorigenesis, ApcMin/+ mice were crossed with the mouse mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) or MMTV-c-Neu transgenic mice. In the PyMT tumor model, the ApcMin/+ mutation significantly decreased survival and tumor latency, promoted a squamous adenocarcinoma phenotype, and enhanced tumor cell proliferation. In tumor-derived cell lines, the proliferative advantage was a result of increased FAK, Src and JNK signaling. These effects were specific to the PyMT model, as no changes were observed in MMTV-c-Neu mice carrying the ApcMin/+ mutation. Our data indicate that heterozygosity of Apc enhances tumor development in an oncogene-specific manner, providing evidence that APC-dependent pathways may be valuable therapeutic targets in breast cancer. Moreover, these preclinical model systems offer a platform for dissection of the molecular mechanisms by which APC mutation enhances breast carcinogenesis, such as altered FAK/Src/JNK signaling

Integrins as therapeutic targets: lessons and opportunities.

The integrins are a large family of cell adhesion molecules that are essential for the regulation of cell growth and function. The identification of key roles for integrins in a diverse range of diseases, including cancer, infection, thrombosis and autoimmune disorders, has revealed their substantial potential as therapeutic targets. However, so far, pharmacological inhibitors for only three integrins have received marketing approval. This article discusses the structure and function of integrins, their roles in disease and the chequered history of the approved integrin antagonists. Recent advances in the understanding of integrin function, ligand interaction and signalling pathways suggest novel strategies for inhibiting integrin function that could help harness their full potential as therapeutic targets