Journal: Breast Cancer Research : BCR

Article Title: Loss of BRCA1 leads to an increase in epidermal growth factor receptor expression in mammary epithelial cells, and epidermal growth factor receptor inhibition prevents estrogen receptor-negative cancers in BRCA1 -mutant mice

doi: 10.1186/bcr2850

Figure Lengend Snippet: BRCA1 suppression in mammary epithelial cells (MECs) leads to an increase in epidermal growth factor receptor (EGFR) expression . (A) MECs were transfected with BRCA1 control or small interfering RNA 1 or 2 (si1, si2), or they were infected with lentivirus-expressing control or BRCA1 -specific small hairpin RNA (sh1, sh2) and lysed for immunoblot analysis.. (B) The intensities of the chemiluminescence signals of EGFR, BRCA1 and tubulin levels were quantified using ImageJ software. (C) Flow cytometry using phycoerythrin-conjugated anti-EGFR antibodies shows an increase in cell surface EGFR expression after BRCA1 suppression (hMEC-hTERT; similar results were obtained with MCF-10A cells). (D) Immunofluorescence of EGFR in asynchronously growing HMLE (top) and after serum deprivation (bottom) in control (left) and BRCA1 -suppressed MECs (right). Experiments were performed in triplicates using controls and two different small hairpin-containing MEC lines. (E) The fluorescence intensity of the images was quantified using ImageJ software.

Article Snippet: MCF-7 cells, the HCC1937 BRCA1 -mutant breast cancer cell line (ATCC) and HCC1937 cells stably transfected with green fluorescent protein (GFP)- BRCA1 (gift from Dr. Ralph Scully) were kept in RPMI 1640 medium with 10% fetal bovine serum.

Techniques: Expressing, Transfection, Small Interfering RNA, Infection, Western Blot, Software, Flow Cytometry, Immunofluorescence, Fluorescence

Journal: Breast Cancer Research : BCR

Article Title: Loss of BRCA1 leads to an increase in epidermal growth factor receptor expression in mammary epithelial cells, and epidermal growth factor receptor inhibition prevents estrogen receptor-negative cancers in BRCA1 -mutant mice

doi: 10.1186/bcr2850

Figure Lengend Snippet: BRCA1 inhibition increases EGFR expression in the aldehyde dehydrogenase 1 (ALDH1)-negative and -positive MEC fractions . Cells were (A, B and C) transfected with either control or BRCA1 small interfering RNA (siRNA) or (D, E and F) infected with lentiviruses expressing control or BRCA1 small hairpin RNA (shRNA). Cells treated with the ALDH1 inhibitor diethylaminobenzaldehyde (DMEA) served as controls (A and D) . (G and H) BRCA1 inhibition increases the expression of cell surface-bound EGFR in human MEC-human telomerase reverse transcriptase (hMEC-hTERT) or MCF-10A cells in the ALDH1-negative and the ALDH1-positive fractions. Live cells were harvested and incubated with ALDH1 reagent followed by immunostaining with phycoerythrin (PE)-conjugated anti-EGFR antibodies and quantification of EGFR using the QuantiBrite standards for PE.

Article Snippet: MCF-7 cells, the HCC1937 BRCA1 -mutant breast cancer cell line (ATCC) and HCC1937 cells stably transfected with green fluorescent protein (GFP)- BRCA1 (gift from Dr. Ralph Scully) were kept in RPMI 1640 medium with 10% fetal bovine serum.

Techniques: Inhibition, Expressing, Transfection, Small Interfering RNA, Infection, shRNA, Incubation, Immunostaining

Journal: Breast Cancer Research : BCR

Article Title: Loss of BRCA1 leads to an increase in epidermal growth factor receptor expression in mammary epithelial cells, and epidermal growth factor receptor inhibition prevents estrogen receptor-negative cancers in BRCA1 -mutant mice

doi: 10.1186/bcr2850

Figure Lengend Snippet: Transcriptional and posttranslational mechnisms lead to an in crease in EGFR expression after BRCA1 inhibition . (A) EGFR mRNA levels were determined in control MECs (light gray bars) and in mammary epithelial cells (MECs) expressing small hairpin RNA directed against BRCA1 (dark bars). RNA levels were normalized for GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression. RT-PCR, real-time reverse transcriptase-polymerase chain reaction; HMLE, human mammary epithelial cells. (B) Decreased EGFR promoter activity as a result of short-term BRCA1 suppression in MECs and MCF-7 cancer cells. BRCA1 control and small interfering RNA (siRNA) and the full-length EGFR promoter were transfected as indicated, and luciferase activity was normalized for Renilla thymidine kinase expression. (C and D) EGFR half-life increases from less than 30 minutes to over 70 minutes after BRCA1 inhibition. Control and BRCA1 sh2-expressing MCF-10A cells were transfected with hemagglutinin-tagged EGFR 48 hours prior to the time course, serum-deprived for 8 hours, then treated with cycloheximide at 100 μg/ml for 2 hours, and stimulated with epidermal growth factor at point 0. Lysates were prepared and immunoblotted at the indicated time points. The chemiluminescence signal was quantified as in Figure 1B. Similar results were obtained with BRCA1 sh1-expressing cells. In D the ratio of the optical density (OD) for EGFR to Tubulin is plotted against time.

Article Snippet: MCF-7 cells, the HCC1937 BRCA1 -mutant breast cancer cell line (ATCC) and HCC1937 cells stably transfected with green fluorescent protein (GFP)- BRCA1 (gift from Dr. Ralph Scully) were kept in RPMI 1640 medium with 10% fetal bovine serum.

Techniques: Expressing, Inhibition, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Activity Assay, Small Interfering RNA, Transfection, Luciferase

Journal: Breast Cancer Research : BCR

Article Title: Loss of BRCA1 leads to an increase in epidermal growth factor receptor expression in mammary epithelial cells, and epidermal growth factor receptor inhibition prevents estrogen receptor-negative cancers in BRCA1 -mutant mice

doi: 10.1186/bcr2850

Figure Lengend Snippet: BRCA1 inhibition leads to increased colony formation and size that is completely blocked by epidermal growth factor receptor (EGFR) inhibition . Mammary epithelial cells (MECs) expressing either (A and B) control small hairpin RNA (shRNA) or BRCA1 -directed shRNA or (C and D) primary MECs isolated from reduction mammoplasties or BRCA1 mutation carriers were seeded and photographed after 10 days in culture. All colony formation was completely suppressed when cells were grown in the presence of erlotinib at 2 μM shown in phase images for each cell type (right). (D) MEC growth is inhibited at concentrations as low as 0.5 μM erlotinib. Cells were seeded at 5,000 cells/well, allowed to grow for 10 days in the presence of the indicated amounts of erlotinib and then counted. (E) Cell viability assay of control and BRCA1 shRNA-expressing human MECs. Cells were seeded in triplicate in 96-well plates at 250 cells/well, and cell viability was determined daily. Dotted line and open symbol represent control cells, and closed symbols represent cells expressing BRCA1-directed shRNA. Black lines, vehicle control; blue lines, culture in the presence of 2.5 μM erlotinib; red line, culture in the presence of 5 μM erlotinib.

Article Snippet: MCF-7 cells, the HCC1937 BRCA1 -mutant breast cancer cell line (ATCC) and HCC1937 cells stably transfected with green fluorescent protein (GFP)- BRCA1 (gift from Dr. Ralph Scully) were kept in RPMI 1640 medium with 10% fetal bovine serum.

Techniques: Inhibition, Expressing, shRNA, Isolation, Mutagenesis, Viability Assay

Journal: Breast Cancer Research : BCR

Article Title: Loss of BRCA1 leads to an increase in epidermal growth factor receptor expression in mammary epithelial cells, and epidermal growth factor receptor inhibition prevents estrogen receptor-negative cancers in BRCA1 -mutant mice

doi: 10.1186/bcr2850

Figure Lengend Snippet: Proliferation and differentiation properties of MECs from MMTV-Cre BRCA1-mutant mice . (A and B) Growth and proliferation properties of mammary epithelial cells (MECs) isolated from mouse mammary tumor virus-Cre recombinase (MMTV-Cre) BRCA1 -mutant mice are similar to MECs isolated from human BRCA1 mutation carriers. MECs were harvested and plated as described previously . (A, top) Cultures from wild-type (WT) control mice resulted in round acinar structures, whereas (A, bottom) cultures from MMTV-Cre BRCA1 flox/flox p53 +/- mice showed complex and irregular features. (B) The overall colony-forming efficiency of murine BRCA1 -mutant MECs is increased. Non-tumor-bearing WT and MMTV- BRCA1 flox/flox p53 +/- MECs were compared at age 7 months, and non-tumor-bearing mice from MMTV-Cre BRCA1 flox/WT (heterozygous loss of BRCA1 ) MMTV-Cre BRCA1 flox/flox were compared at age 12 to 13 months. (C) Mammary glands from MMTV-Cre BRCA1 flox/flox p53 +/- mice contain epidermal growth factor receptor (EGFR) and aldehyde dehydrogenase 1 (ALDH1)-positive acini. Immunohistochemistry for EGFR and ALDH1 was performed on five BRCA1 -mutant mammary glands and seven Cre-negative controls. Representative images are shown at × 20 original magnification. (D) Erlotinib is active in suppressing the growth of murine MECs from mice of WT, MMTV-Cre BRCA1 flox/flox or MMTV BRCA1 flox/flox p53 +/- background. All MECs were seeded in Matrigel-based cultures and photographed and analyzed for colony formation using SIGNATURE software after 14 days of culture.

Article Snippet: MCF-7 cells, the HCC1937 BRCA1 -mutant breast cancer cell line (ATCC) and HCC1937 cells stably transfected with green fluorescent protein (GFP)- BRCA1 (gift from Dr. Ralph Scully) were kept in RPMI 1640 medium with 10% fetal bovine serum.

Techniques: Mutagenesis, Isolation, Immunohistochemistry, Software

Journal: Breast Cancer Research : BCR

Article Title: Loss of BRCA1 leads to an increase in epidermal growth factor receptor expression in mammary epithelial cells, and epidermal growth factor receptor inhibition prevents estrogen receptor-negative cancers in BRCA1 -mutant mice

doi: 10.1186/bcr2850

Figure Lengend Snippet: Erlotinib as a chemopreventative agent in (MMTV-Cre) BRCA1 flox/flox p53 +/- mice . (A) Erlotinib prevents the emergence of estrogen receptor-negative (ER - ) but not estrogen receptor-positive (ER + ) breast cancers in mouse mammary tumor virus-Cre recombinase (MMTV-Cre) BRCA1 flox/flox p53 +/- mice. Virgin female mice were treated as controls or with 100 mg/kg erlotinib via oral gavage once daily, seven days per week. Tumors were recorded when they were first palpated. Kaplan-Meier graphing and analysis of disease-free survival were performed using the GraphPad Prism software package. (B) The growth of established breast cancers is not affected by erlotinib treatment. Mice that developed tumors in the control cohort were switched to erlotinib treatment, and the tumor volume relative to the tumor volume at diagnosis was plotted against treatment time. ER status was determined after necropsy. The trend line for vehicle control-treated tumors was established on the basis of the tumor volumes of control mice.

Article Snippet: MCF-7 cells, the HCC1937 BRCA1 -mutant breast cancer cell line (ATCC) and HCC1937 cells stably transfected with green fluorescent protein (GFP)- BRCA1 (gift from Dr. Ralph Scully) were kept in RPMI 1640 medium with 10% fetal bovine serum.

Techniques: Software

Journal: BioMed Research International

Article Title: RNA Sequencing Analysis Reveals Interactions between Breast Cancer or Melanoma Cells and the Tissue Microenvironment during Brain Metastasis

doi: 10.1155/2017/8032910

Figure Lengend Snippet: Xenograft models of brain metastasis and primary breast cancer. (a) For models of brain metastasis, human breast cancer (231-Luc, HER2-60, and HER2-90) or melanoma (MeWo, WM3734) cell lines (or PBS as a control) were injected into the left ventricle of female immunodeficient mice. For models of primary breast cancer, human 231-Luc or HCC1937 cells were orthotopically injected into mammary fat pads. (b) Hematoxylin-eosin staining of mouse brain tissue with metastases formed by 231-Luc, MeWo, or WM3734 cells. Arrows indicate human metastatic cancer cells. Scale bars, 200 μ m.

Article Snippet: Human breast cancer cell line HCC1937 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS.

Techniques: Injection, Staining

Journal: International Journal of Medical Sciences

Article Title: Antioxydation And Cell Migration Genes Are Identified as Potential Therapeutic Targets in Basal-Like and BRCA1 Mutated Breast Cancer Cell Lines

doi: 10.7150/ijms.20508

Figure Lengend Snippet: Main characteristics of the cell lines.

Article Snippet: MDA-MB-231 and HCC1937 human breast cancer cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and were grown in RPMI medium supplemented with 10% foetal calf serum, 2 mM L-glutamine and 20 μg/ml gentamicin.

Techniques: Mutagenesis

Journal: International Journal of Medical Sciences

Article Title: Antioxydation And Cell Migration Genes Are Identified as Potential Therapeutic Targets in Basal-Like and BRCA1 Mutated Breast Cancer Cell Lines

doi: 10.7150/ijms.20508

Figure Lengend Snippet: Summary of transcriptome and methylome data.

Article Snippet: MDA-MB-231 and HCC1937 human breast cancer cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and were grown in RPMI medium supplemented with 10% foetal calf serum, 2 mM L-glutamine and 20 μg/ml gentamicin.

Techniques: Standard Deviation

Journal: International Journal of Medical Sciences

Article Title: Antioxydation And Cell Migration Genes Are Identified as Potential Therapeutic Targets in Basal-Like and BRCA1 Mutated Breast Cancer Cell Lines

doi: 10.7150/ijms.20508

Figure Lengend Snippet: Comparison of RNA-Seq and immunohistochemistry data.

Article Snippet: MDA-MB-231 and HCC1937 human breast cancer cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and were grown in RPMI medium supplemented with 10% foetal calf serum, 2 mM L-glutamine and 20 μg/ml gentamicin.

Techniques: Immunohistochemistry

Journal: International Journal of Medical Sciences

Article Title: Antioxydation And Cell Migration Genes Are Identified as Potential Therapeutic Targets in Basal-Like and BRCA1 Mutated Breast Cancer Cell Lines

doi: 10.7150/ijms.20508

Figure Lengend Snippet: List of the 77 genes highly expressed and significantly up-regulated in basal-like breast cancer cell lines.

Article Snippet: MDA-MB-231 and HCC1937 human breast cancer cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and were grown in RPMI medium supplemented with 10% foetal calf serum, 2 mM L-glutamine and 20 μg/ml gentamicin.

Techniques:

Journal: iScience

Article Title: BRCA1 mediates protein homeostasis through the ubiquitination of PERK and IRE1

doi: 10.1016/j.isci.2022.105626

Figure Lengend Snippet: Depleting UPR components is lethal to the BRCA1-def cancer cells (A) MDA-MB-436 (BRCA1-def or +), HCC1937 BRCA1-def and SUMPT149 BRCA1-def breast cancer cells were transfected with control, CypB, IRE1, GRP94, HSP70, BiP, PERK or DERL1 siRNA and assessed for cell survival. (B) HCC1937 BRCA1+, MDA-MB-231, MCF7, and MCF10A cells were transfected with CypB siRNA and assessed for cell survival. (C) MDA-MB-436 (+ or def) and HCC1937 (+ or def) breast cancer cells were transfected with control, CypB-A or CypB-B siRNA. Top, western blot analysis. Bottom, clonal cell survival analysis. (D) MDA-MB-436 BRCA1-def cells were transfected with either (i) pCMV vector and siRNA control, (ii) PPIB/pCMV and siRNA control, (iii) pCMV control and CypB 3′UTR siRNA or (iv) PPIB/pCMV and CypB 3′UTR siRNA for cell survival and Western blot analysis. N.S. = not significant. F-CypB = Flag-tagged cyclophilin B. Bar charts are the summary of the quantitative analysis of the colony forming units (CFU) from each siRNA transfection experiment. Data are displayed as mean ± SD. P values were calculated by Student’s two-tailed, unpaired t -test. See also Figures S7 and .

Article Snippet: HCC1937 human breast cancer cells (ATCC) were cultured in RPMI1640 medium (SH3002701, Life Technologies) supplemented with 10% fetal bovine serum and 1% pen/strep.

Techniques: Transfection, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: BRCA1 mediates protein homeostasis through the ubiquitination of PERK and IRE1

doi: 10.1016/j.isci.2022.105626

Figure Lengend Snippet: Cyclophilin inhibitors induce severe synthetic lethality in BRCA1-def cancer cells in vitro and in vivo (A) BRCA1-def cancer cells (MDA-MB-436, HCC1937, SUM149PT or UWB1.289), BRCA1-proficient cancer cells (MDA-MB-436, MDA-MB-231 or MCF7) and non-tumorigenic mammary epithelial cells (MCF10A) were seeded one day prior to drug treatment. A single continuous dose of CsA, NIM811 or Alisporivir was added to the corresponding cell lines. Quantitative analysis of each survival curve is shown in graphs. CsA = Cyclosporin A. CFU = Colony forming units. DMSO = dimethyl sulfoxide vehicle. (B) CsA treatment of human BRCA1-def breast cancer cells in a murine model. Representative luciferase images of each treated subject were taken by an In Vivo Imaging systems (IVIS). Animals were either treated with vehicle (control) or CsA (treatment) for 6 weeks post-tumor formation. (C) Quantitative analysis of tumor progression from each group was summarized in graph. (D) Schematic diagram depicts the model of wildtype and mutant BRCA1 regulation of the ERAD and UPR signaling pathways to maintain protein homeostasis in the ER. Wt = wildtype. def = deficient. Ub = ubiquitin. P = phosphate. Dashed arrow = less favored pathway. Ubiquitination and phosphorylation modifications in the diagram are simplified for clarity. Data are displayed as mean ± SD. P values were calculated by Student’s two-tailed, unpaired t -test.

Article Snippet: HCC1937 human breast cancer cells (ATCC) were cultured in RPMI1640 medium (SH3002701, Life Technologies) supplemented with 10% fetal bovine serum and 1% pen/strep.

Techniques: In Vitro, In Vivo, Luciferase, In Vivo Imaging, Mutagenesis, Two Tailed Test

Journal: Cancers

Article Title: Eribulin and Paclitaxel Differentially Alter Extracellular Vesicles and Their Cargo from Triple-Negative Breast Cancer Cells

doi: 10.3390/cancers13112783

Figure Lengend Snippet: Effects of eribulin or paclitaxel on CD63 localization in HCC1937 cells. HCC1937 cells were treated with vehicle (VEH), 50 nM eribulin (ERB), or 500 nM paclitaxel (PTX) for 4 h to cause maximal microtubule disruption and then co-immunostained for CD63 (green) and β-tubulin (red). ( A ) Immunofluorescence images are representative of two experiments with equal exposures across conditions. Scale bar = 25 µm. ( B ) Quantification of CD63 regional intensity and ( C ) CD63 spots analysis using Operetta™ high-content imaging. Mean cellular fluorescence for a mean of 432 cells (range: 109–1265) was calculated for n = 9 wells and represents three independent experiments. Significance determined by one-way ANOVA with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: MDA-MB-231 and HCC1937 triple-negative breast cancer (TNBC) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Immunofluorescence, Imaging, Fluorescence

Journal: Cancers

Article Title: Eribulin and Paclitaxel Differentially Alter Extracellular Vesicles and Their Cargo from Triple-Negative Breast Cancer Cells

doi: 10.3390/cancers13112783

Figure Lengend Snippet: Effects of eribulin and paclitaxel on sEV-associated proteins. MDA-MB-231 and HCC1937 cells were treated for 8 h with vehicle, 25 nM eribulin, or 50 nM paclitaxel and sEVs were isolated from the conditioned media. ( A , B ) Representative immunoblots of indicated proteins in 10 µg of cell lysates and equal volumes of sEVs representing 7.5% (CD63, CD9, GAPDH) or 42% (calnexin, CD81, flotillin-1) of the yield of 1.2 × 10 8 MDA-MB-231 cells ( A ) or 6–8 × 10 7 HCC1937 cells ( B ). ( C – F ) Band intensities for indicated proteins in sEV samples normalized to vehicle control sEVs. Points represent the results of independent experiments and bars represent the mean ± SEM, n = 4–6. Significance determined by one-way ANOVA with Tukey’s post-hoc. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: MDA-MB-231 and HCC1937 triple-negative breast cancer (TNBC) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Isolation, Western Blot

Journal: Cancers

Article Title: Eribulin and Paclitaxel Differentially Alter Extracellular Vesicles and Their Cargo from Triple-Negative Breast Cancer Cells

doi: 10.3390/cancers13112783

Figure Lengend Snippet: Fluorescent nanoparticle tracking analysis of CD63 positive sEVs. MDA-MB-231 ( A – D ) or HCC1937 ( E – H ) cells were treated for 8 h with vehicle, 25 nM eribulin, or 50 nM paclitaxel and sEVs collected from the conditioned media. Samples were labeled with anti-CD63-AF488 fluorescent antibodies to quantify CD63+ sEVs and NTA performed with the ZetaView™ in fluorescent mode to measure CD63+ particles. ( A , E ) Overlay of NTA size distribution vs. concentration graphs for each treatment measuring CD63+ sEVs. ( B , F ) The mean CD63+ particle concentration, ( C , G ) percent CD63+ particles, determined by dividing the CD63+ concentration by the total sEV concentration for each sample, and ( D , H ) median CD63+ sEV diameters for each treatment are shown. Points in the bar graphs represent the results of independent experiments and the bars represent the mean ± SEM, n = 4–6, and significance determined by one-way ANOVA with Tukey’s post-hoc test, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: MDA-MB-231 and HCC1937 triple-negative breast cancer (TNBC) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Labeling, Concentration Assay

Journal: Cancers

Article Title: Eribulin and Paclitaxel Differentially Alter Extracellular Vesicles and Their Cargo from Triple-Negative Breast Cancer Cells

doi: 10.3390/cancers13112783

Figure Lengend Snippet: Effects of eribulin or paclitaxel on sEV-associated ILK and β1-integrin. HCC1937 cells were treated for 8 h with vehicle, 25 nM eribulin, or 50 nM paclitaxel and sEVs were isolated from the conditioned media. ( A ) Representative immunoblots of the indicated proteins in 10 µg of cell lysates and equal volumes of sEVs representing 42% (ILK, β1-integrin, flotillin-1) or 7.5% (GAPDH) of the yield from 6–8 × 10 7 HCC1937 cells. ( B ) Band intensities for indicated proteins in sEV samples normalized to vehicle control sEVs. Points represent the results of independent experiments and bars the mean ± SEM, n = 3–4. Significance was determined among sEV samples by one-way ANOVA with Tukey’s post-hoc test, * p < 0.05 and ** p < 0.01.

Article Snippet: MDA-MB-231 and HCC1937 triple-negative breast cancer (TNBC) cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Isolation, Western Blot