Journal: Nature Communications

Article Title: The oncogene AAMDC links PI3K-AKT-mTOR signaling with metabolic reprograming in estrogen receptor-positive breast cancer

doi: 10.1038/s41467-021-22101-7

Figure Lengend Snippet: a Analysis of somatic alterations of AAMDC using cancer genomic data sets and tools available from cBioPortal (see “Methods”). The frequency of amplification is shown as a percentage and the sample numbers are shown in brackets. METABRIC Molecular Taxonomy of Breast Cancer International Consortium, TCGA The Cancer Genome Atlas, BRCA Breast Cancer, INSERM Institut national de la santé et de la recherche médicale, MBC Metastatic Breast Cancer, NSCLC non-small-cell lung carcinoma, FHCRC Fred Hutchinson Cancer Research Center, NEPC National Environment Protection Council, PanCan Pan-Cancer. b Kaplan–Meier survival plots for patients with tumors expressing high (red) or low (green) levels of AAMDC mRNA. The lower left plots correspond to luminal B tumors treated with tamoxifen (see “Methods”). The p value shown for each plot is determined by the log-rank test. GEO Gene Expression Omnibus, GSE genomic spatial event, NSCLC non-small-cell lung carcinoma. c Localization of the AAMDC protein in tumors from a breast tissue microarray (TMA) assessed by immunohistochemistry (IHC). Representative IHC sections of normal breast tissue, estrogen receptor-negative (ER − ) tumor tissue, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) are shown. 0, 1+, 2+, 3+ indicate the staining intensity score. d Associations between AAMDC expression (IHC) and lymph node metastasis (LN + ) as well as tumor grade, tumor size (T3-4), and ER positivity (ER + ) by AAMDC localization from the same TMA. Statistical significance is indicated by Chi-square analysis with a one-tailed p -value relative to ER − tissue. For T3-4: * p = 0.03; for LN + : * p = 0.03; for ER + , from left to right: * p = 0.003, * p = 0.005, * p = 0.005. n = 60 biologically independent samples. Full details of the TMA are provided in Supplementary Table . e Frequency of AAMDC amplification/polysomy in a cohort of 119 luminal B breast cancer specimens. Representative fluorescence in situ hybridization (FISH) images are indicated, with specific probes for AAMDC (red) and Centromere enumeration 11 probe for chromosome 11 ( C11 , green). The full clinical and pathological features of these tumors are shown in Supplementary Data . f Real-time expression analyses (qRT-PCR) of AAMDC in luminal, non-luminal, and normal-like breast cells. Significance levels are determined relative to MCF-12A by Ordinary one-way ANOVA with Dunnett multiple comparison test. Data are presented as mean values ± SEM (* p = 0.0217, ** p = 0.0018, **** p < 0.0001). n = 3 biologically independent RNA extractions. Representative images of immunocytochemistry (ICC) and FISH of selected luminal cell lines are presented. HuMECs non-transformed human mammary epithelial cells.

Article Snippet: For experiments involving non-genetically manipulated human mammary epithelial cells (HuMECs) (Fig. ), human primary mammary epithelial cells were purchased from ATCC (HMEC, PCS-600-010).

Techniques: Amplification, Expressing, Gene Expression, Microarray, Immunohistochemistry, In Situ, Staining, One-tailed Test, Fluorescence, In Situ Hybridization, Quantitative RT-PCR, Comparison, Immunocytochemistry, Transformation Assay

Journal: Cancers

Article Title: Human Breast Extracellular Matrix Microstructures and Protein Hydrogel 3D Cultures of Mammary Epithelial Cells

doi: 10.3390/cancers13225857

Figure Lengend Snippet: Histopathological features of normal and invasive ductal carcinoma tissues and ECM. ( a ) H&E staining (upper panels) of the cross sections of the normal breast tissue ( i ), decellularized normal breast tissue ECM ( ii ), breast IDC tissue ( iii ), and decellularized breast IDC tissue ECM ( iv ) as well as IF staining (bottom panels) of the corresponding tissue or ECM sections ( v – viii ). Green stain, Col I; blue stain (Hoechst), nuclei; white stain, Ki-67. Arrows indicate the normal or cancerous acini or ECM structures. Insets magnify the indicated regions of interest. Scale bars: ( i – iv , vi , viii ), 20 μm; ( ii , iv ) insets, 10 μm; ( v , vii ), 100 μm; ( vi , viii ) insets, 50 μm. ( b ) High magnification of IF images showing the acinar structures void of Col I staining of the breast epithelial cells (yellow arrows) in normal breast tissues ( i ) and positive for the infiltrated cancer cells (yellow arrows) within the acini lumens in IDC tissues ( ii ). Green stain, Col I; blue stain (Hoechst), nuclei; white stain, Ki-67. Scale bars: ( i , ii ), 20 μm. ( c ) Graphed Young’s modulus values for the elasticities of the tissues or ECM measured with AFM. A minimum of seven measuring spots on three consecutive cross sections of each sample were used for data acquisition. Representative microscopic images of tip-probing status were exhibited on top of the values of the individual samples. Error bars, standard deviation (SD).

Article Snippet: Normal primary human mammary epithelial cells (HUMEC, ScienCell Research Laboratories, Carlsbad, CA, USA) were grown in Mammary Epithelial Cell Medium (ScienCell Research Laboratories).

Techniques: Staining, Standard Deviation

Journal: Cancers

Article Title: Human Breast Extracellular Matrix Microstructures and Protein Hydrogel 3D Cultures of Mammary Epithelial Cells

doi: 10.3390/cancers13225857

Figure Lengend Snippet: HB-TMG support of human breast epithelial cell 3D growth. ( a ) MCF10A cell suspending growth within HB-TMG in the presence or absence of E2 or/and IGF-I stimulation. Arrows, cell spheres. Scale bars, 50 μm. ( b ) Quantification of the volumes (vol) of the Day 12 MCF10A cell 3D aggregates shown in ( a ). * p < 0.05; ** p < 0.01. ( c ) IF staining of normal breast or breast cancer epithelial cell culture on 2D surfaces or 3D HB-TMG. Arrows, cell protrusions. Red, phalloidin; green, Col I; blue, nucleus Hoechst staining. Scale bars, 5 μm. ( d ) Quantification of cell protrusions in 2D cultures. * p < 0.05; ** p < 0.01. ( e ) Quantification of cell protrusions in 3D cultures. ( f ) IF staining of acini formation of normal breast or breast cancer epithelial cells. Red, β4 integrin; green, E-cadherin; blue, nucleus Hoechst staining. Scale bars: c-i, c-ii, and c-iv, 20 μm; c-iii, 50 μm.

Article Snippet: Normal primary human mammary epithelial cells (HUMEC, ScienCell Research Laboratories, Carlsbad, CA, USA) were grown in Mammary Epithelial Cell Medium (ScienCell Research Laboratories).

Techniques: Staining, Cell Culture

Journal: Breast Cancer Research : BCR

Article Title: Distinct phenotypes of cancer cells on tissue matrix gel

doi: 10.1186/s13058-020-01321-7

Figure Lengend Snippet: Mammary epithelial cell metabolic changes and response to lactate depletion on the hydrogels. a , b NMR spectroscopy data of major metabolite changes in the culture media of HUMEC or MM231 cells grown on the three types of hydrogels for 7 days. HRMAS spectra, blue = decoupled proton spectra; red = 13 C- 1 H coupled spectra. c – h Effect of lactate depletion from the culture medium on the proliferation of HUMEC or the different subtypes of breast cancer cells grown on Matrigel. Error bars represent mean ± SD of three independent experiments, with each experiment having triplicate samples. There were no significant differences in the proliferation of the HUMEC cells cultured on the different gels. MM231 cells: * P < 0.05 for 0.05 U/ml versus 0.2 or 0.8 U/ml ( t = 18.172 and t = 19.492) and 0.05 U/ml versus no LOX ( t = 15.280), respectively, and ** P < 0.001 for 0.2 U/ml and 0.8 U/ml versus no LOX ( t = 30.454 and t = 26.801). There was no significant difference between the 0.2 U/ml and 0.8 U/ml groups; MM468 cells: * P < 0.05 for 0.05 U/ml versus 0.2 and 0.8 U/ml ( t = 7.268 and t = 4.577), ** P < 0.001 for no LOX versus 0.05, 0.2, and 0.8 U/ml ( t = 6.865, t = 8.387, and t = 9.639); T47D cells: * P < 0.05 for no LOX versus 0.05, 0.2, and 0.8 U/ml ( t = 26.869, t = 6.242, and t = 3.380). There was no significant difference between the 0.05, 0.2, and 0.8 U/ml groups; BT474 cells: * P < 0.05 for 0.8 U/ml versus 0.05 and 0.2 U/ml ( t = 6.038 and t = 5.201), ** P < 0.001 for 0.05, 0.2, and 0.8 U/ml versus no LOX ( t = 7.388, t = 9.115, and t = 12.747). There was no significant difference between the 0.05 U/ml and 0.2 U/ml groups; SKBR3 cells: * P < 0.05 or ** P < 0.001, comparing all concentrations of LOX except for between 0.05 U/ml and 0.2 U/ml, which had no significant difference (no LOX vs. 0.05, 0.2, and 0.8 U/ml, t = 3.807, t = 2.817, and t = 7.523, respectively; 0.8 U/ml vs. 0.05 and 0.2 U/ml, t = 2.655 and t = 3.7587)

Article Snippet: MDA-MB-231 (MM231), MDA-MB-468 (MM468), T47D, BT474, and SKBR3 breast cancer cells and MCF10A normal human mammary epithelial cells were from ATCC; normal primary human mammary epithelial cells (HUMEC) were from ScienCell.

Techniques: Spectroscopy, Cell Culture

Journal: Scientific Reports

Article Title: Arsenic hexoxide has differential effects on cell proliferation and genome-wide gene expression in human primary mammary epithelial and MCF7 cells

doi: 10.1038/s41598-021-82551-3

Figure Lengend Snippet: Proposed model of AS6 effects in normal mammary epithelial cells vs MCF7 cells. ( A ) In our study, it is shown that AS6 over 2 μM is toxic to both HUMEC and MCF7 cells to induce apoptotic cell death. By contrast, AS6 has cytotoxicity selectively to MCF7 cells, but not in HUMEC, at milder concentrations below 1 μM. Pinkish hexamers, AS6; rounded cells, apoptotic cells; upward arrow, upregulation; downward arrow; downregulation. ( B ) Proposed mode of action of AS6 (AS 4 O 6 ). AS6 at milder concentrations might effectively target cancer cells to apoptotic cell death without much affecting normal cells. AS6 impairs the synthesis of key DNA repair enzymes for homologous recombination, which leads to genome instability. A potent CDKs/cyclins inhibitor p21 is activated to arrest the cell cycle while cell stress responses are provoked to facilitate the cell decision to apoptosis.

Article Snippet: Primary mammary normal epithelial cells (HUMEC, ATCC PCS-600-010) and MCF7 (ATCC HTB-22) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Homologous Recombination