Journal: Human Gene Therapy

Article Title: LyP-1-Modified Oncolytic Adenoviruses Targeting Transforming Growth Factor β Inhibit Tumor Growth and Metastases and Augment Immune Checkpoint Inhibitor Therapy in Breast Cancer Mouse Models

doi: 10.1089/hum.2020.078

Figure Lengend Snippet: LyP-1 receptor (p32) and/or TGFβ-1 expression in breast cancer cell lines and patients. (A) LyP-1 receptor (p32) expression in human and mouse breast cancer cells by immunofluorescence staining. In MDA-MB-231 and 4T1 cells, LyP-1 receptors were localized more on plasma membrane (white arrows) and endoplasmic reticulum-like membrane networks (white triangular arrowheads). In MCF-7 cells, they were more enriched in Golgi-like juxtanuclear compartments (white arrows). Scale bar = 25 μm. (B) LyP-1 receptor expression in breast cancer cell lines by Western blot. Note that mouse LyP-1 receptors in 4T1 cells have a slightly different banding pattern from those in human breast cancer cells. All experiments were repeated three times. (C) Representative images of LyP-1 receptor and TGFβ-1 expression in breast cancer patient samples. Yellow arrows point to positive p32 IHC staining in tumor cells; red open arrows point to TGFβ-1 staining in tumor stroma. Magnification is indicated by scale bars. IHC, immunohistochemistry; TGFβ-1, transforming growth factor β-1.

Article Snippet: Human mammary tumor cell lines, MCF-7 (ATCC, Manassas, VA), MDA-MB-231 (ATCC), and MDA-MB-231-luc2, 16 and the mouse mammary tumor cell line, 4T1 (ATCC), were maintained as described earlier.

Techniques: Expressing, Immunofluorescence, Staining, Clinical Proteomics, Membrane, Western Blot, Immunohistochemistry

Journal: Molecular cancer research : MCR

Article Title: Leptin Signaling Mediates Obesity-associated CSC Enrichment and EMT in Preclinical TNBC Models

doi: 10.1158/1541-7786.MCR-17-0508

Figure Lengend Snippet: CSC/EMT markers are elevated in tumors from DIO mice in association with increased local leptin signaling. (a) Tumor expression of several CSC/EMT-related genes in DIO and control mice identified by RNA sequencing analysis was verified via quantitative RT-PCR. (b) Tumor expression in DIO and control mice of previously established CSC/EMT-related genes (11) not identified by RNA sequencing was measured by quantitative RT-PCR. (c) ALDH activity, another marker of CSC enrichment, was quantified in DIO and control mouse tumors. (d) Immunohistochemical staining for tumor E-cadherin and vimentin expression in DIO and control mice. Representative images shown at x20 magnification. (e) Tumor expression of Lepr as well as mammary fat pad (MFP) expression of Lep in DIO and control mice were measured via quantitative RT-PCR. *P<0.05, **P<0.01

Article Snippet: Cell lines Two mouse mammary tumor cell lines isolated from MMTV-Wnt-1 mice in 2010 by the Hursting laboratory and found to cluster with the basal-like (E-Wnt) and claudin-low (M-Wnt) breast cancer subtypes (11), and human MDA-MB-231 breast cancer cells (ATCC #HTB-26, obtained in 2011), which cluster with the claudin-low subtype, were used in in vitro studies.

Techniques: Expressing, Control, RNA Sequencing, Quantitative RT-PCR, Activity Assay, Marker, Immunohistochemical staining, Staining

Journal: Molecular cancer research : MCR

Article Title: Leptin Signaling Mediates Obesity-associated CSC Enrichment and EMT in Preclinical TNBC Models

doi: 10.1158/1541-7786.MCR-17-0508

Figure Lengend Snippet: Leptin stimulates mammosphere formation in triple-negative mammary tumor cells. Mammosphere formation in (a) E-Wnt, (b) M-Wnt, and (c) MDA-MB-231 cells was assessed at the end of propagation 1 (P1), during which the cells were treated for 7 days with leptin, and following propagation 2 (P2), in which the spheres from P1 were dissociated and then replated with the same treatments for another 7 days. Representative images of mammospheres at the end of P2 are shown at x10 magnification. (d) Lepr expression in parental E-Wnt cells (EWnt-P) as well as E-Wnt cells stably transfected with a scrambled shRNA plasmid (EWnt-S) or shRNA to Lepr (EWnt-L1 and EWnt-L2) was measured by quantitative RT-PCR. (e) Mammosphere formation was assessed in EWnt-P, EWnt-S, EWnt-L1, and EWnt-L2 cells after a 7-day incubation in mammosphere media. Socs3 (f) and Foxc2, Twist2, and Vim (g) gene expression was measured by quantitative RT-PCR. Different letters indicate significant differences, P<0.05.

Article Snippet: Cell lines Two mouse mammary tumor cell lines isolated from MMTV-Wnt-1 mice in 2010 by the Hursting laboratory and found to cluster with the basal-like (E-Wnt) and claudin-low (M-Wnt) breast cancer subtypes (11), and human MDA-MB-231 breast cancer cells (ATCC #HTB-26, obtained in 2011), which cluster with the claudin-low subtype, were used in in vitro studies.

Techniques: Expressing, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Quantitative RT-PCR, Incubation, Gene Expression

Journal: Molecular cancer research : MCR

Article Title: Leptin Signaling Mediates Obesity-associated CSC Enrichment and EMT in Preclinical TNBC Models

doi: 10.1158/1541-7786.MCR-17-0508

Figure Lengend Snippet: Obesity-associated circulating factors promote triple-negative mammary tumor cell viability, migration, invasion, and a CSC/EMT genotype. (a) E-Wnt, M-Wnt, and MDA-MB-231 cell viability following a 48-hour exposure to media containing 2% DIO or control mouse serum was assessed by MTT assay. (b) Migration of E-Wnt, M-Wnt, and MDA-MB-231 cells during a 6-hour exposure to media containing 2% DIO or 2% control mouse serum was measured by wound healing assay. Representative images of cells at baseline and 6 hours are shown at x10 magnification. (c) The invasive capacity of E-Wnt, M-Wnt, and MDA-MB-231 cells in response to chemoattraction with media containing 2% DIO or 2% control mouse serum over 24 hours was measured using Matrigel invasion chambers. Representative images of invading cells are shown at x10 magnification. (d) Expression of CSC/EMT-related genes in E-Wnt, M-Wnt, and MDA-MB-231 cells following a 24-hour exposure to media containing 2% DIO or 2% control mouse serum was measured by quantitative RT-PCR. *P<0.05, **P<0.01, ***P<0.001

Article Snippet: Cell lines Two mouse mammary tumor cell lines isolated from MMTV-Wnt-1 mice in 2010 by the Hursting laboratory and found to cluster with the basal-like (E-Wnt) and claudin-low (M-Wnt) breast cancer subtypes (11), and human MDA-MB-231 breast cancer cells (ATCC #HTB-26, obtained in 2011), which cluster with the claudin-low subtype, were used in in vitro studies.

Techniques: Migration, Control, MTT Assay, Wound Healing Assay, Expressing, Quantitative RT-PCR

Journal: Molecular cancer research : MCR

Article Title: Leptin Signaling Mediates Obesity-associated CSC Enrichment and EMT in Preclinical TNBC Models

doi: 10.1158/1541-7786.MCR-17-0508

Figure Lengend Snippet: Proposed model illustrating leptin-mediated upregulation in CSC/EMT-related genes and phenotype. Our findings suggest that obesity in MMTV-Wnt-1 mice promotes both an excess of leptin production in the tumor microenvironment (normal mammary tissue) and an upregulation in tumor expression of the leptin receptor and CSC/EMT-related genes. They also indicate that leptin signaling promotes a CSC/EMT-related phenotype, including increased CSC enrichment and cell viability, migration, and invasion, and specifically regulates the expression of Foxc2, Twist2, Vim, Akt3, and Sox2 in triple-negative mammary tumor cells. We hypothesize that these genes may mediate the observed leptin-induced CSC/EMT-related phenotype and that leptin regulates these genes via stimulation of the JAK2/STAT3 and/or PI3K/Akt pathways. Black arrows indicate effects observed in this study, solid blue arrows indicate pathways known from the literature, and dotted blue arrows indicate hypothesized mechanisms.

Article Snippet: Cell lines Two mouse mammary tumor cell lines isolated from MMTV-Wnt-1 mice in 2010 by the Hursting laboratory and found to cluster with the basal-like (E-Wnt) and claudin-low (M-Wnt) breast cancer subtypes (11), and human MDA-MB-231 breast cancer cells (ATCC #HTB-26, obtained in 2011), which cluster with the claudin-low subtype, were used in in vitro studies.

Techniques: Expressing, Migration

Journal: Glia

Article Title: Endoplasmic Reticulum Stress Amplifies Cytokine Responses in Astrocytes via a PERK / eIF2α / JAK1 Signaling Axis

doi: 10.1002/glia.70067

Figure Lengend Snippet: JAK1 and PERK are required to augment TNF‐α induced gene expression in human glioma cells. (A) 1321N1 cells stably expressing Cas9 were transfected with non‐targeting (NT) or JAK1 guide RNAs (gRNA) to establish non‐clonal cell lines. These cells were then treated with IFN‐γ (10 ng/mL) for 30 min followed by immunoblotting. (B) NT and JAK1 gRNA (#2) cells were treated with thaps (1 μM), TNF‐α (10 ng/mL), or both for 4 h followed by qPCR. (C) 1321N1 cells stably expressing Cas9 were transfected with NT or PERK gRNA to establish non‐clonal cell lines. These cells were then treated with thaps (1 μM) for the indicated times followed by immunoblotting. (D) NT and PERK gRNA cells were treated with thaps (1 μM), TNF‐α (10 ng/mL), or both for 4 h followed by qPCR. N = 4, data are means ± standard deviation.

Article Snippet: 1321N1 human astrocytoma cell line stably expressing Cas9 nuclease was purchased from GeneCopoeia.

Techniques: Gene Expression, Stable Transfection, Expressing, Transfection, Western Blot, Standard Deviation

Journal: NPJ Breast Cancer

Article Title: NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients

doi: 10.1038/s41523-019-0106-x

Figure Lengend Snippet: NDRG4 depletion in breast tumor cells increases lymph node adhesion and cell migration toward VN. a NDRG4 expression meta-analysis in human breast cancer cell lines using GOBO application . Breast cancer subtypes can be identified by different colors: red = Basal A, gray = Basal B and blue = Luminal cells. b Analysis of NDRG4 mRNA expression levels in MCF-7 cells transfected with NDRG4-shRNAs or scramble control (shSCR). Expression levels are relative to wild type cells and normalized to hydroxymethylbilane synthase (HMBS) gene. Error bars represent SEM of biological replicates ( n = 5). *** p < 0.001, ns = not significant, by one-way ANOVA. c Western blot analysis of NDRG4 expression in cytoplasmic extracts of MCF-7 shNDRG4s or shSCRs cells, using antibodies against NDRG4 and Actin. MCF-7 cells express all the three isoforms of NDRG4: 37 kDa (NDRG4-B), 39 kDa (NDRG4-B var ) and 41 kDa isoform (NDRG4-H). For simplicity, results obtained for two independent shNDRG4 clones were grouped and presented as the shNDRG4 group. ( d , left) Representative images of adherent red fluorescent MCF-7 cells on frozen rat lymph node sections. d , e Quantification of adherent MCF7 ( d ) and T47D ( e ) breast tumor cells in lymph nodes sections (right, n = 4 independent experiments for MCF-7 and 3 for T47D, ** p < 0.01 by two-tailed t test). f Stationary adhesion assays showed that NDRG4 knockdown promotes ‘adhesive switch’ between FN and VN. Error bars represent SEM of biological replicates ( n = 4). Cells do not adhere to albumin control (BSA, data not shown). * p < 0.01, ** p < 0.01, ns = not significant by two-tailed t tests. g , h Haptotactic cell migration toward VN was analyzed by transwell migration assay in MCF-7 ( g ) and T47D ( h ) cell lines. Error bars represent SEM of biological replicates ( n = 4). *** p < 0.001 by two-tailed t tests

Article Snippet: For stable transfection of NDRG4 shRNA into MCF-7 and T47D breast tumor cells, the pGFP-V-RS vector containing shRNA inserts which targeted the 3′-unttranslated region of NDRG4 (TRCN0000134583 and TRCN0000137216) was purchased (Origene).

Techniques: Migration, Expressing, Transfection, Western Blot, Clone Assay, Two Tailed Test, Transwell Migration Assay

Journal: NPJ Breast Cancer

Article Title: NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients

doi: 10.1038/s41523-019-0106-x

Figure Lengend Snippet: NDRG4 knockdown promotes clustering of β1-integrin at the leading edge of T47D cells. Representative confocal images of β1 integrin subunit (MAB1965, green) at the ventral cell surface of VN-adherent T47D shNDRG4 or shSCR cells

Article Snippet: For stable transfection of NDRG4 shRNA into MCF-7 and T47D breast tumor cells, the pGFP-V-RS vector containing shRNA inserts which targeted the 3′-unttranslated region of NDRG4 (TRCN0000134583 and TRCN0000137216) was purchased (Origene).

Techniques: