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:

Journal: The Journal of steroid biochemistry and molecular biology

Article Title: Antiestrogens in combination with immune checkpoint inhibitors in breast cancer immunotherapy

doi: 10.1016/j.jsbmb.2019.105415

Figure Lengend Snippet: Biologic activity of selected SERD candidates. A) Downregulation of ER protein. ER-positive MCF-7 cells were treated in phenol-red free RPMI 1640 without FBS and containing vehicle control (CON) or 100 nM concentrations of either fulvestrant (FX) or antiestrogens 105, 109, 121, 140, 151, 160 or JD128 in vitro. After 4 hours, cells were harvested and processed for PAGE and Western immunoblots using ERα antibody (1D5, Thermofisher Scientific). RPL13A was used as a loading control. B) Specific [3H]estradiol-17β (E2) binding and competition for binding by antiestrogen JD128 or fulvestrant (FX) at 10 nM was assessed in human MCF-7 breast cancer cells using methods as described before [36, 41]. C) Response of the ERE-luciferase T47D reporter construct to estrogen antagonists fulvestrant (10 nM) or JD128 (10 nM) in combination with 2nM 17β-estradiol as compared to treatment with 17β-estradiol alone (E2; 2 nM). Cells were dosed with either E2 alone or with SERDs combined with E2 in phenol red-free medium with 0.1% dextran-coated charcoal-treated FBS in luminometer plates. Data are presented as relative light units (RLU) relative to that of E2 alone in three replicate assays (4 wells per replicate) + SEM. Treatment with E2 alone induced a 12-fold induction of ER-dependent luciferase activity quantified as RLU relative to vehicle control-treated samples.

Article Snippet: A stable ER-positive T47D ERE luciferase reporter cell line, in which the ERE and the reporter luciferase gene are consistently expressed in the cell line were used in this study (Signosis).

Techniques: Activity Assay, Control, In Vitro, Western Blot, Binding Assay, Luciferase, Construct

Journal: The Journal of steroid biochemistry and molecular biology

Article Title: Antiestrogens in combination with immune checkpoint inhibitors in breast cancer immunotherapy

doi: 10.1016/j.jsbmb.2019.105415

Figure Lengend Snippet: Steroid-like SERD 128 inhibits estrogen-induced BC cell proliferation in vitro and in vivo. A) ER-positive MCF-7, T47D and ZR75 cells were grown in phenol red-free media with 1% DCC-FBS for 48 hr., then treated 48 hr. with 2 nM estradiol-17β alone (control) or in combination with 10 nM doses of JD128. Note that MCF-7 cell populations included cells with no HER2-overexpression (MCF-7/PAR), cells with HER2-overexpression (MCF-7/HER2) and MCF-7 cells with tamoxifen resistance (MCF-7/TMR). Cell proliferation is shown as % of that in estradiol-treated controls (n=3 experiments). Inhibition of cell proliferation in MCF-7/PAR, MCF-7/HER2, MCF-7/TMR, T47D and ZR75 cells averaged 98%, 85%, 94%, 97% and 98% as compared to estradiol-treated controls. JD128 significantly blocked proliferation in all BC cell models in vitro (P<0.001). Of note, E2 alone stimulated cell proliferation several-fold in each cell line as compared to cells treated only with vehicle (not shown). B) JD128 inhibits growth of human breast tumor xenografts in vivo. MCF-7 human breast cancer cells were subcutaneously inoculated in nude mice previously primed with estradiol pellets. When animals developed tumors of comparable size they were randomized to treatment with vehicle control (control) or JD128 at 15 and 75 mg/kg once a day by oral gavage for 28 days. Tumors were measured every 3 days, and tumor volume was calculated as V= (l ×w × w)/2). Results are expressed as mean ± SEM. *** P < 0.001 as compared to control group.

Article Snippet: A stable ER-positive T47D ERE luciferase reporter cell line, in which the ERE and the reporter luciferase gene are consistently expressed in the cell line were used in this study (Signosis).

Techniques: In Vitro, In Vivo, Control, Over Expression, Inhibition