Journal: American Journal of Cancer Research

Article Title: Upregulation of CYP1B1 by hypoxia is mediated by ERα activation in breast cancer cells

doi:

Figure Lengend Snippet: Analysis of HIF-1α and CYP1B1 expression in tissue microarray (TMA) samples from breast cancer patients. (A) Schematic diagram of the tumor stages of each breast tissue core. (B) Hematoxylin and eosin (H&E) staining images provided by the manufacturer. Immunofluorescence images of tumor microarrays (TMA) stained with anti-hypoxia-inducible factor-1 alpha (HIF-1α) (C) and anti-cytochrome P450 1B1 (CYP1B1) (D) antibodies. Images were acquired at 20X magnification using a Leica Thunder microscope. Whole tissue slide images were mosaic merged via LAS-X software. (E) Representative images of immunofluorescence analysis of HIF-1α (green) and CYP1B1 (green) in two slides of ER-negative and positive breast cancer TMA. Immunofluorescence images were acquired using a Leica Thunder microscope. (F) Scatter plots showing fluorescence intensity correlation analysis of HIF-1α and CYP1B1 in 64 ER-positive TMAs. (G) Violin plots of fluorescence intensities of HIF-1α and CYP1B1. Samples were classified into low (immunohistochemical [IHC] score: 1+, 2+) and high (IHC score: 3+) groups according to the estrogen receptor (ER) expression status of the TMA specification sheet, and the correlation between HIF-1α and CYP1B1 expression was analyzed. (H) Analysis of fluorescence intensity of CYP1B1 against HIF1α expression in ER-positive TMA. The fluorescence intensity of CYP1B1 was analyzed by classifying the fluorescence intensity of HIF1-α based on the top and bottom 25% ER expression. *, P<0.0001 versus ER-positive tumor tissue with low HIF1-α fluorescence intensity by Mann-Whitney test.

Article Snippet: Immunofluorescence analysis of tumor microarrays (TMAs) A human breast cancer tissue microarray (TMA, #BC081120e) containing 110 cases was purchased from Biomax (Rockville, MD).

Techniques: Expressing, Microarray, Staining, Immunofluorescence, Microscopy, Software, Fluorescence, Immunohistochemical staining, MANN-WHITNEY

Journal: Molecular cancer research : MCR

Article Title: Suppression of Breast Cancer Stem Cells and Tumor Growth by the RUNX1 Transcription Factor

doi: 10.1158/1541-7786.MCR-18-0135

Figure Lengend Snippet: (A) Representative tissue microarray images of RUNX1 in normal breast tissue. (B) and (C) Representative tissue microarray images of RUNX1 in breast tumor tissues. Red circle: malignant regions; Blue circle: normal glandular tissues. (D) Kaplan-Meier analysis showed higher overall survival in patients with higher RUNX1 mRNA expression (GSE37751, GSE7390 and TCGA). Gehan-Breslow-Wilcoxon test with p value<0.01, p value<0.05, p value<0.01 respectively, comparing patients with high RUNX1 expression and low RUNX1 expression in three data sets.

Article Snippet: Tissue microarray Tissue microarray data of RUNX1 in breast cancer patients were obtained from Human Protein Atlas ( www.proteinatlas.org ) [ 40 ].

Techniques: Microarray, Expressing

Journal: Molecular cancer research : MCR

Article Title: Suppression of Breast Cancer Stem Cells and Tumor Growth by the RUNX1 Transcription Factor

doi: 10.1158/1541-7786.MCR-18-0135

Figure Lengend Snippet: (A) MCF10CA1a cells were injected into the mammary fat pad of SCID mice. Points represent mean tumor volume. (B) Western blot analyses show RUNX1 and E-cadherin levels are decreased, and Vimentin level is increased in tumor samples compared to MCF10CA1a cells. (C) Protein quantification show that RUNX1 is significant decreased in tumor samples compared to MCF10CA1a cells (Upper). RT-qPCR analyses of RNA from tumor samples show decreased human RUNX1 expression of compared with MCF10CA1a cells. Mouse embryonic fibroblast (MEF) is used as the negative control as human Runx1 is not detected in those samples (Lower). Student’s t test * p value <0.05, *** p value <0.001 and. Error bars represent the standard error of the mean (SEM) from three independent experiments. (D) RT-qPCR analyses of RNA from tumor samples show activation of mesenchymal marks Vimentin and FN1 and other tumor growth related genes including MMP9, MMP13, VGF, CXCR4 and CXCL12 compared with MCF10CA1a cells. The primers of these genes are human specific as they are tested negative in mouse embryonic fibroblast. Student’s t test * p value <0.05, ** p value <0.01, *** p value <0.001 and **** p value <0.0001. Error bars represent the standard error of the mean (SEM) from three independent experiments.

Article Snippet: Tissue microarray Tissue microarray data of RUNX1 in breast cancer patients were obtained from Human Protein Atlas ( www.proteinatlas.org ) [ 40 ].

Techniques: Injection, Western Blot, Quantitative RT-PCR, Expressing, Negative Control, Activation Assay

Journal: Molecular cancer research : MCR

Article Title: Suppression of Breast Cancer Stem Cells and Tumor Growth by the RUNX1 Transcription Factor

doi: 10.1158/1541-7786.MCR-18-0135

Figure Lengend Snippet: (A) Western blot analyses confirm RUNX1 overexpression in MCF10AT1 (Upper) and MCF10CA1a (Lower) cells. Vimentin expression is repressed upon RUNX1 overexpression in both cell lines. (B) Representative phase contrast images (magnification 100×) of MCF10AT1 and MCF10CA1a cells with EV control or RUNX1 overexpression subjected to a scratch assay for times indicated. The area of the scratch was plotted as a percentage of total area for N = 3 independent experiments carried out in duplicate. (C) Light microscopy images (mag. 12×) of stained cells from a representative (1 of N = 2) trans-well migration assay experiment MCF10AT1 and MCF10CA1a cells with EV control or RUNX1 overexpression (left); quantitation of migrated cells assessed by measurement of the absorbance of solubilized crystal violet stain retained by migrated cells (right). (D) Light microscopy images (mag. 12×) of stained cells from a representative (1 of N= 2) trans-well matrigel invasion assay experiment with MCF10AT1 and MCF10CA1a cells with EV control or RUNX1 overexpression to evaluate invasion (left); quantitation of invaded cells assessed by measurement of the absorbance of solubilized crystal violet stain retained by invaded cells (right). For all assays, three independent experiments were carried out in duplicates. All quantitative data are depicted as mean ± S.E. per group. *P < 0.05, **P < 0.01 (student’s t-test).

Article Snippet: Tissue microarray Tissue microarray data of RUNX1 in breast cancer patients were obtained from Human Protein Atlas ( www.proteinatlas.org ) [ 40 ].

Techniques: Western Blot, Over Expression, Expressing, Control, Wound Healing Assay, Light Microscopy, Staining, Migration, Quantitation Assay, Invasion Assay

Journal: Molecular cancer research : MCR

Article Title: Suppression of Breast Cancer Stem Cells and Tumor Growth by the RUNX1 Transcription Factor

doi: 10.1158/1541-7786.MCR-18-0135

Figure Lengend Snippet: (A) A total of 1 × 106 MCF10CA1a cells with EV or RUNX1 overexpression were injected into mammary fat pad of SCID mice (n = 7 in each group). The points represent average tumor volume at each time point ± SD. P values were obtained by 2-tailed Student t test. *, P < 0.05; ***, P<0.001; ****, P<0.0001. (B) Tumor size measured at day 28 (end point). P values were obtained by 2-tailed Student t test. *, P < 0.05. (C) Tumor weight at day 28 (end point). P values were obtained by 2-tailed Student t test. *, P < 0.05. (D) Representative luminescence images at 4 weeks after mammary fat pad injection.

Article Snippet: Tissue microarray Tissue microarray data of RUNX1 in breast cancer patients were obtained from Human Protein Atlas ( www.proteinatlas.org ) [ 40 ].

Techniques: Over Expression, Injection

Journal: Molecular cancer research : MCR

Article Title: Suppression of Breast Cancer Stem Cells and Tumor Growth by the RUNX1 Transcription Factor

doi: 10.1158/1541-7786.MCR-18-0135

Figure Lengend Snippet: (A) Western blot analyses show RUNX1 is decreased and Zeb1, Twist1 and Vimentin level are increased in BCSC samples compared to Parental and Bulk MCF10AT1 cells. Right, protein quantification shows that RUNX1 is significant decreased in BCSC. (B) Tumorsphere formation efficiency for BCSC populations is significantly higher than bulk population. **P < 0.01. (C) RUNX1 overexpression in MCF10CA1a cells reduces tumorsphere formation efficiency. *P < 0.05. Right, represent picture of tumorsphere. (D) RUNX1 overexpression in MCF10AT1 cells reduces tumorsphere formation efficiency. *P < 0.05 Right, represent picture of tumorsphere. (E) Western blot analyses of lysates from MCF10AT1 cells treated with shRUNX1 show decreased protein expression of RUNX1 and E-cadherin and increased protein expression of Vimentin. (F) RUNX1 knockdown in MCF10AT1 cells activates tumorsphere formation efficiency. *P < 0.05. Right, represents picture of tumorsphere.

Article Snippet: Tissue microarray Tissue microarray data of RUNX1 in breast cancer patients were obtained from Human Protein Atlas ( www.proteinatlas.org ) [ 40 ].

Techniques: Western Blot, Over Expression, Expressing, Knockdown

Journal: Molecular cancer research : MCR

Article Title: Suppression of Breast Cancer Stem Cells and Tumor Growth by the RUNX1 Transcription Factor

doi: 10.1158/1541-7786.MCR-18-0135

Figure Lengend Snippet: (A) Flow cytometric analysis of CD44 and CD24 expression in MCF10AT1 cells with EV or RUNX1 overexpression. (B) Flow cytometric analysis of CD44 and CD24 expression in MCF10AT1 cells stably expressing RUNX1 or non-silencing shRNAs.

Article Snippet: Tissue microarray Tissue microarray data of RUNX1 in breast cancer patients were obtained from Human Protein Atlas ( www.proteinatlas.org ) [ 40 ].

Techniques: Expressing, Over Expression, Stable Transfection

Journal: Molecular cancer research : MCR

Article Title: Suppression of Breast Cancer Stem Cells and Tumor Growth by the RUNX1 Transcription Factor

doi: 10.1158/1541-7786.MCR-18-0135

Figure Lengend Snippet: (A) Western blot analyses show Zeb1 is decreased upon RUNX1 overexpression in MCF10AT1 cells. (B) Western blot analyses show Zeb1 is activated upon RUNX1 knockdown in MCF10AT1 cells. (C) ChIP-qPCR confirmation of RUNX1 occupancy at Zeb1. RUNX1 binding is increased in RUNX1 overexpression samples. Data obtained with antibodies against RUNX1 are normalized to input control and ZNF188 (NC1) and ZNF333 (NC2), which were used as the negative control as RUNX1 are predicted not to bind these genes. (D) Western blot analyses show Zeb1 knockdown by siRNA in MCF10AT1 RUNX1 depleted cells. (E) Flow cytometric analysis of CD44 and CD24 expression in MCF10AT1 cells RUNX1 and Zeb1 double knockdown cells. *P<0.05 (F) Zeb1 knockdown in MCF10AT1 RUNX1 depleted cells reduces tumorsphere formation efficiency. ***P < 0.001 (G) Mechanism on how RUNX1 represses tumor growth in breast cancer. (EC-epithelial like cells; MC-mesenchymal-like cells).

Article Snippet: Tissue microarray Tissue microarray data of RUNX1 in breast cancer patients were obtained from Human Protein Atlas ( www.proteinatlas.org ) [ 40 ].

Techniques: Western Blot, Over Expression, Knockdown, ChIP-qPCR, Binding Assay, Control, Negative Control, Expressing