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Park S.,Lunenfeld Tanenbaum Research Institute at Mount Sinai Hospital
British Journal of Cancer | Year: 2017

Background:VEPH1 is amplified in several cancers including ovarian but its impact on tumour progression is unknown. Previous work has shown that VEPH1 inhibits TGFβ signalling while its Drosophila ortholog increases tissue growth, raising the possibility that VEPH1 could impact tumour growth or progression.Methods:A CRISPR approach was used to disrupt VEPH1 expression in ovarian cancer ES-2 cells, while VEPH1-negative SKOV3 cells were stably transfected with VEPH1 cDNA. The impact of altered VEPH1 expression was assessed using in vitro and in vivo assays and mechanistic studies were performed in vitro.Results:VEPH1 expression in SKOV3 cells resulted in a reduced tumour growth rate associated with increased necrotic area, and decreased microvessel density and VEGF-A levels relative to tumours formed by mock-transfected cells. VEPH1 expression also decreased VEGFA and IL8 expression in SKOV3 cells and was associated with decreased activated AKT levels. These effects were not observed in ES-2 cells, which bear a BRAFV600E activating mutation that leads to constitutively increased IL8 and VEGFA expression.Conclusions:VEPH1 expression in SKOV3 ovarian cancer cells inhibits AKT activation to decrease VEGFA and IL8 expression, which leads to decreased tumour vascularisation and progression.British Journal of Cancer advance online publication: 16 March 2017;10.1038/bjc.2017.51 www.bjcancer.com. © 2017 Cancer Research UK


Teo G.,National University of Singapore | Liu G.,Mount Sinai Hospital | Zhang J.,Mount Sinai Hospital | Nesvizhskii A.I.,University of Michigan | And 3 more authors.
Journal of Proteomics | Year: 2014

Significance Analysis of INTeractome (SAINT) is a statistical method for probabilistically scoring protein-protein interaction data from affinity purification-mass spectrometry (AP-MS) experiments. The utility of the software has been demonstrated in many protein-protein interaction mapping studies, yet the extensive testing also revealed some practical drawbacks. In this paper, we present a new implementation, SAINTexpress, with simpler statistical model and quicker scoring algorithm, leading to significant improvements in computational speed and sensitivity of scoring. SAINTexpress also incorporates external interaction data to compute supplemental topology-based scores to improve the likelihood of identifying co-purifying protein complexes in a probabilistically objective manner. Overall, these changes are expected to improve the performance and user experience of SAINT across various types of high quality datasets. Biological significance: We present SAINTexpress, an upgraded implementation of Significance Analysis of INTeractome (SAINT) for filtering high confidence interaction data from affinity purification-mass spectrometry (AP-MS) experiments. SAINTexpress features faster computation and incorporation of external data sources into the scoring, improving the performance and user experience of SAINT across various types of datasets. © 2013 Elsevier B.V.


Zhang L.,Jilin University | Zhang L.,Ontario Cancer Institute | Yang Z.,Jilin University | Yang Z.,Ontario Cancer Institute | And 9 more authors.
Oncotarget | Year: 2016

We employed a high-throughput drug library screening platform to identify novel agents affecting thyroid cancer cells. We used human thyroid cancer cell lines to screen a collection of approximately 5200 small molecules with biological and/or pharmacologial properties. Parallel primary screens yielded a number of hits differentially active between thyroid and melanoma cells. Amongst compounds specifically targeting thyroid cancer cells, colchicine emerged as an effective candidate. Colchicine inhibited cell growth which correlated with G2 cell cycle arrest and apoptosis. These effects were hampered through inhibition of MEK1/2 and JNK. In contrast, inhibition of p38-MAPK had little effect, and AKT had no impact on colchicine action. Systemic colchicine inhibited thyroid cancer progression in xenografted mice. These findings demonstrate that our screening platform is an effective vehicle for drug reposition and show that colchicine warrants further attention in well-defined clinical niches such as thyroid cancer.


Couzens A.L.,Lunenfeld Tanenbaum Research Institute at Mount Sinai Hospital | Knight J.D.R.,Lunenfeld Tanenbaum Research Institute at Mount Sinai Hospital | Kean M.J.,Lunenfeld Tanenbaum Research Institute at Mount Sinai Hospital | Kean M.J.,University of Toronto | And 15 more authors.
Science Signaling | Year: 2013

The Hippo pathway regulates organ size and tissue homeostasis in response to multiple stimuli, including cell density and mechanotransduction. Pharmacological inhibition of phosphatases can also stimulate Hippo signaling in cell culture. We defined the Hippo protein-protein interaction network with and without inhibition of serine and threonine phosphatases by okadaic acid. We identified 749 protein interactions, including 599 previously unrecognized interactions, and demonstrated that several interactions with serine and threonine phosphatases were phosphorylation-dependent. Mutation of the T-loop of MST2 (mammalian STE20-like protein kinase 2), which prevented autophosphorylation, disrupted its association with STRIPAK (striatin- interacting phosphatase and kinase complex). Deletion of the aminoterminal forkhead-associated domain of SLMAP (sarcolemmal membrane-associated protein), a component of the STRIPAK complex, prevented its association with MST1 and MST2. Phosphatase inhibition produced temporally distinct changes in proteins that interacted with MOB1A and MOB1B (Mps one binder kinase activator-like 1A and 1B) and promoted interactions with upstream Hippo pathway proteins, such as MST1 and MST2, and with the trimeric protein phosphatase 6 complex (PP6). Mutation of three basic amino acids that are part of a phospho-serine- and phospho-threonine-binding domain in human MOB1B prevented its interaction with MST1 and PP6 in cells treated with okadaic acid. Collectively, our results indicated that changes in phosphorylation orchestrate interactions between kinases and phosphatases in Hippo signaling, providing a putative mechanism for pathway regulation.


Liu J.C.,Toronto General Research Institute | Voisin V.,University of Toronto | Wang S.,Toronto General Research Institute | Wang S.,University of Toronto | And 17 more authors.
EMBO Molecular Medicine | Year: 2014

The tumor suppressors Pten and p53 are frequently lost in breast cancer, yet the consequences of their combined inactivation are poorly understood. Here, we show that mammary-specific deletion of Pten via WAP-Cre, which targets alveolar progenitors, induced tumors with shortened latency compared to those induced by MMTV-Cre, which targets basal/luminal progenitors. Combined Pten-p53 mutations accelerated formation of claudin-low, triple-negative-like breast cancer (TNBC) that exhibited hyper-activated AKT signaling and more mesenchymal features relative to Pten or p53 single-mutant tumors. Twenty-four genes that were significantly and differentially expressed between WAP-Cre:Pten/p53 and MMTV-Cre:Pten/p53 tumors predicted poor survival for claudin-low patients. Kinome screens identified eukaryotic elongation factor-2 kinase (eEF2K) inhibitors as more potent than PI3K/AKT/mTOR inhibitors on both mouse and human Pten/p53-deficient TNBC cells. Sensitivity to eEF2K inhibition correlated with AKT pathway activity. eEF2K monotherapy suppressed growth of Pten/p53-deficient TNBC xenografts in vivo and cooperated with doxorubicin to efficiently kill tumor cells in vitro. Our results identify a prognostic signature for claudin-low patients and provide a rationale for using eEF2K inhibitors for treatment of TNBC with elevated AKT signaling. © 2014 The Authors.


Salem O.,Lady Davis Institute for Medical Research | Wang H.T.,Lady Davis Institute for Medical Research | Alaseem A.M.,Lady Davis Institute for Medical Research | Ciobanu O.,Lady Davis Institute for Medical Research | And 9 more authors.
Arthritis Research and Therapy | Year: 2014

Introduction: We previously showed that type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification), is constitutively expressed by mesenchymal stem cells (MSCs) from osteoarthritis patients and this may be related to Naproxen (Npx), a nonsteroidal anti-inflammatory drug used for therapy. Hedgehog (HH) signaling plays an important role during the development of bone. We tested the hypothesis that Npx affected osteogenic differentiation of human MSCs through the expression of Indian hedgehog (IHH), Patched-1 (PTC1) and GLI family members GLI1, GLI2, GLI3 in vitro.Methods: MSCs were cultured in osteogenic differentiation medium without (control) or with 0.5 μM Npx. The expression of collagen type X, alpha 1 (COL10A1), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OC), collagen type I, alpha 1 (COL1A1) was analyzed with real-time reverse transcription (RT) PCR, and the ALP activity was measured. The osteogenesis of MSCs was monitored by mineral staining and quantification with alizarin red S. To examine whether Npx affects osteogenic differentiation through HH signaling, the effect of Npx on the expression of IHH, GLI1, GLI2, GLI3 and PTC1 was analyzed with real-time RT PCR. The effect of cyclopamine (Cpn), a HH signaling inhibitor, on the expression of COL10A1, ALP, OC and COL1A1 was also determined.Results: When MSCs were cultured in osteogenic differentiation medium, Npx supplementation led to a significant decrease in ALP gene expression as well as its activity, and had a tendency to decrease mineral deposition. It also decreased the expression of COL1A1 significantly. In contrast, the gene expression of COL10A1 and OPN were upregulated significantly by Npx. No significant effect was found on OC expression. The expression of IHH, PTC1, GLI1, and GLI2 was increased by Npx, while no significant difference was observed on GLI3 expression. Cpn reversed the effect of Npx on the expression of COL10A1, ALP, OPN and COL1A1.Conclusions: These results indicate that Npx can affect gene expression during osteogenic differentiation of MSCs, and downregulate mineral deposition in the extracellular matrix through IHH signaling. Therefore, Npx could affect MSC-mediated repair of subchondral bone in OA patients. © 2014 Salem et al.; licensee BioMed Central Ltd.


PubMed | University of Toronto, Lunenfeld Tanenbaum Research Institute at Mount Sinai Hospital and Cell and Systems Biology and.
Type: Journal Article | Journal: Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

Drosophila melted encodes a pleckstrin homology (PH) domain-containing protein that enables normal tissue growth, metabolism, and photoreceptor differentiation by modulating Forkhead box O (FOXO), target of rapamycin, and Hippo signaling pathways. Ventricular zone expressed PH domain-containing 1 (VEPH1) is the mammalian ortholog of melted, and although it exhibits tissue-restricted expression during mouse development and is potentially amplified in several human cancers, little is known of its function. Here we explore the impact of VEPH1 expression in ovarian cancer cells by gene-expression profiling. In cells with elevated VEPH1 expression, transcriptional programs associated with metabolism and FOXO and Hippo signaling were affected, analogous to what has been reported for Melted. We also observed altered regulation of multiple transforming growth factor- (TGF-) target genes. Global profiling revealed that elevated VEPH1 expression suppressed TGF--induced transcriptional responses. This inhibitory effect was verified on selected TGF- target genes and by reporter gene assays in multiple cell lines. We further demonstrated that VEPH1 interacts with TGF- receptor I (TRI) and inhibits nuclear accumulation of activated Sma- and Mad-related protein 2 (SMAD2). We identified two TRI-interacting regions (TIRs) with opposing effects on TGF- signaling. TIR1, located at the N terminus, inhibits canonical TGF- signaling and promotes SMAD2 retention at TRI, similar to full-length VEPH1. In contrast, TIR2, located at the C-terminal region encompassing the PH domain, decreases SMAD2 retention at TRI and enhances TGF- signaling. Our studies indicate that VEPH1 inhibits TGF- signaling by impeding the release of activated SMAD2 from TRI and may modulate TGF- signaling during development and cancer initiation or progression.


PubMed | Lunenfeld Tanenbaum Research Institute at Mount Sinai Hospital
Type: Journal Article | Journal: Science signaling | Year: 2013

The Hippo pathway regulates organ size and tissue homeostasis in response to multiple stimuli, including cell density and mechanotransduction. Pharmacological inhibition of phosphatases can also stimulate Hippo signaling in cell culture. We defined the Hippo protein-protein interaction network with and without inhibition of serine and threonine phosphatases by okadaic acid. We identified 749 protein interactions, including 599 previously unrecognized interactions, and demonstrated that several interactions with serine and threonine phosphatases were phosphorylation-dependent. Mutation of the T-loop of MST2 (mammalian STE20-like protein kinase 2), which prevented autophosphorylation, disrupted its association with STRIPAK (striatin-interacting phosphatase and kinase complex). Deletion of the amino-terminal forkhead-associated domain of SLMAP (sarcolemmal membrane-associated protein), a component of the STRIPAK complex, prevented its association with MST1 and MST2. Phosphatase inhibition produced temporally distinct changes in proteins that interacted with MOB1A and MOB1B (Mps one binder kinase activator-like 1A and 1B) and promoted interactions with upstream Hippo pathway proteins, such as MST1 and MST2, and with the trimeric protein phosphatase 6 complex (PP6). Mutation of three basic amino acids that are part of a phospho-serine- and phospho-threonine-binding domain in human MOB1B prevented its interaction with MST1 and PP6 in cells treated with okadaic acid. Collectively, our results indicated that changes in phosphorylation orchestrate interactions between kinases and phosphatases in Hippo signaling, providing a putative mechanism for pathway regulation.

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