Journal: Carcinogenesis

Article Title: miR-200a inhibits migration of triple-negative breast cancer cells through direct repression of the EPHA2 oncogene

doi: 10.1093/carcin/bgv087

Figure Lengend Snippet: EPHA2 is correlated with poor clinical outcome in TNBC and is a predicted target of miR-200a. (A) Kaplan–Meier survival plots show that high level of EPHA2 mRNA is significantly correlated to a lower overall survival in a cohort of basal-like patients (N = 343). This correlation is not significant for Luminal B (N = 495), or HER2-positive (N = 111) subtype patients. For Luminal A (N = 651) patients, high levels of EPHA2 correlate with better survival. (B) A potential target site for miR-200a was identified in the 3′UTR sequence of EPHA2 at nucleotides 716–737, using target prediction software TargetScan. Eight nucleotides within the seed sequence of miR-200a showed complete complementarity with the 3′UTR sequence of EPHA2. These nucleotides were 100% conserved between various vertebrate species. (C) Western blotting of undifferentiated HC11 cells transfected with miR-200a mimic (25nM) or control in a single (left), or double transfection (right), shows decreased levels of EPHA2 protein by miR-200a. β-Actin was used as a loading control. (D) Significant reductions of EPHA2 mRNA levels were noted within 24h after miR-200a mimic (25nM) treatment in MDA-MB-231 and SUM159 cells, compared to control mimic. (E) Western blotting of MDA-MB-231 and SUM159 cells, 48h after transfection with miR-200a mimic (25nM) or control mimic, shows that miR-200a reduced expression of EPHA2 protein in both cell lines. β-Actin was used as a loading control. Student’s t-test, two-tailed distribution was performed and P ≤ 0.05 was considered statistically significant.

Article Snippet: To overexpress EPHA2, 800ng of EPHA2 open reading frame (ORF) expression clone and negative control vector (both from Genecopoeia, Rockville, MD, USA) were transfected using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer instruction.

Techniques: Sequencing, Software, Western Blot, Transfection, Control, Expressing, Two Tailed Test

Journal: Carcinogenesis

Article Title: miR-200a inhibits migration of triple-negative breast cancer cells through direct repression of the EPHA2 oncogene

doi: 10.1093/carcin/bgv087

Figure Lengend Snippet: miR-200a directly targets the 3′UTR of EPHA2. (A) The constructs contain EPHA2 3′UTR sequence encompassing the predicted miR-200a site or control with (a) this site mutated, and (b) no 3′UTR sequence, cloned into luciferase reporter vectors. The constructs contains hLuc and hRLuc under the control of Sv40 and CMV promoter. hRLuc is endogenous control of construct expression. (B) HEK239 cells were transfected with plasmid containing WT or mutated EPHA2 3′UTR, or control plasmid, and either miR-200a mimic, control mimic or miR-9 mimic. Luciferase activity was measured 24h post transfection. Only the construct containing the predicted target site significantly decreased its luciferase activity, and only when miR-200a was added. (C) EPHA2 mRNA levels were reduced also in HEK239 cells. The cells were transfected with miR-200a mimic (25nM) or control mimic and collected 24h post transfection. EPHA2 and housekeeping gene 36B4 mRNA levels were measured using qPCR and data analyzed using the ΔΔCT method. For significance, Student’s t-test, two-tailed distribution was used.

Article Snippet: To overexpress EPHA2, 800ng of EPHA2 open reading frame (ORF) expression clone and negative control vector (both from Genecopoeia, Rockville, MD, USA) were transfected using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer instruction.

Techniques: Construct, Sequencing, Control, Clone Assay, Luciferase, Expressing, Transfection, Plasmid Preparation, Activity Assay, Two Tailed Test

Journal: Carcinogenesis

Article Title: miR-200a inhibits migration of triple-negative breast cancer cells through direct repression of the EPHA2 oncogene

doi: 10.1093/carcin/bgv087

Figure Lengend Snippet: miR-200a reduces migration of TNBC cells through EPHA2 regulation. (A) The effect of miR-200a and EPHA2 on migration was measured in MDA-MB-231 and SUM159 cells. Cells transfected with miR-200a mimic (50nM), si-EPHA2 (100nM), EPHA2-ORF (800ng) or corresponding control for 48h were subjected to wound-healing assay. Images were taken at 0 and 12h (or 9h) after wound introduction. The white line highlights the wound edge at 0 and 12h (or 9h). (B) Quantification of relative cell migration in MDA-MB-231 and SUM159 transfected as shown in A. Distance of gap closure was measured using ImageJ. Data are presented as mean ± SD from two independent experiments. Unpaired two-tailed student’s t-test was used for significant testing, and presented as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (C) Overexpression of EPHA2 rescued the anti-migratory effect of miR-200a in MDA-MB-231 and SUM159 cells. Cells were cotransfected with miR-200a mimic and EPHA2-ORF or control ORF and subjected to wound-healing assay.

Article Snippet: To overexpress EPHA2, 800ng of EPHA2 open reading frame (ORF) expression clone and negative control vector (both from Genecopoeia, Rockville, MD, USA) were transfected using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer instruction.

Techniques: Migration, Transfection, Control, Wound Healing Assay, Two Tailed Test, Over Expression

Journal: Carcinogenesis

Article Title: miR-200a inhibits migration of triple-negative breast cancer cells through direct repression of the EPHA2 oncogene

doi: 10.1093/carcin/bgv087

Figure Lengend Snippet: EPHA2 does not affect E-cadherin levels but increases AMPK phosphorylation. (A) E-cadherin levels are independent on EPHA2 in TNBC cells. MDA-MB-231 and SUM159 cells were transfected with miR-200a mimic (50nM), s-iEPHA2 or corresponding control were subjected to western blot for E-cadherin. β-Actin was used as a loading control. (B) AMPK phosphorylation is enhanced by miR-200a-EPHA2 pathway. MDA-MB-231 cells were transfected and followed by western blot for pAMPK and total AMPK. (C) Inhibition of AMPK promotes cell migration in TNBC cells. Cells were treated with Compound C for 8h followed by wound-healing assay. Images were taken at 0 and 12h after wound introduction and relative migration distance was quantified using ImageJ.

Article Snippet: To overexpress EPHA2, 800ng of EPHA2 open reading frame (ORF) expression clone and negative control vector (both from Genecopoeia, Rockville, MD, USA) were transfected using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer instruction.

Techniques: Phospho-proteomics, Transfection, Control, Western Blot, Inhibition, Migration, Wound Healing Assay

Journal: Carcinogenesis

Article Title: miR-200a inhibits migration of triple-negative breast cancer cells through direct repression of the EPHA2 oncogene

doi: 10.1093/carcin/bgv087

Figure Lengend Snippet: Model illustrating how miR-200a regulates TNBC migration. (A) Differentiated epithelial cells expressing E-cadherin, EPHA2 and Ephrin. (B) TNBC cells exhibit downregulation of E-cadherin and lack of Ephrin along with a migratory and invasive phenotype. (C) miR-200a regulates both the E-cadherin and EPHA2 pathways, resulting in increased levels of E-cadherin and reduced levels of EPHA2 along with inhibited migratory and invasive capacities. (D) Proposed pathways that miR-200a can repress migration through dual pathways: by direct targeting the 3′UTR of EPHA2 (solid lines, as demonstrated in this study) and through the indirect upregulation of the E-cadherin (dashed lines, as reported in literature).

Article Snippet: To overexpress EPHA2, 800ng of EPHA2 open reading frame (ORF) expression clone and negative control vector (both from Genecopoeia, Rockville, MD, USA) were transfected using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer instruction.

Techniques: Migration, Expressing

Journal: Oncogene

Article Title: Targeting EphA2 impairs cell cycle progression and growth of basal-like/triple-negative breast cancers

doi: 10.1038/onc.2017.170

Figure Lengend Snippet: Clinical relevance of EphA2 in basal-like/triple-negative breast cancer. ( a ) Heat map analysis of EPHA2 and EFNA1 messenger RNA expression in breast cancer microarray data from The Cancer Genome Atlas (TCGA) stratified by PAM50 molecular subtype revealed EPHA2 enrichment in the basal-like subtype concomitant with mutually exclusive EFNA1 expression. ( b ) Kaplan–Meier analysis of data set with microarray profiles from 316 ER − /PR − human basal-like breast cancer samples (KMplot.com). Upper tertile expressing highest EPHA2 levels (red line) significantly correlated with lower recurrence-free (RF) survival probability. ( c ) Analysis of human TNBC tissue microarray (TMA) revealed high-level EphA2 protein expression in 65% of samples (31 out of 48 positive/high) relative to 20% of control (normal/hyperplastic and benign fibroadenoma; 2 out of 10 positive/high) samples from previously analyzed human breast cancer TMA. HR, hazard ratio.

Article Snippet: For EphA2 expression analyses, EphA2 protein expression in TNBC cores were compared to expression in normal/hyperplastic and benign fibroadenoma control samples from Cybrdi TMA CC08-10-001.

Techniques: RNA Expression, Microarray, Expressing, Control

Journal: Oncogene

Article Title: Targeting EphA2 impairs cell cycle progression and growth of basal-like/triple-negative breast cancers

doi: 10.1038/onc.2017.170

Figure Lengend Snippet: EphA2 knockdown impairs proliferation in human TNBC cell lines. ( a ) Growth of TNBC lines stably expressing lentiviral control vector or EphA2 shRNA (EphA2 KD) was assessed by MTT assay. Loss of EphA2 expression significantly impaired growth in MDA-MB-231, BT549, HCC1395, HCC1806 and HCC1937 lines (* P <0.05, Student’s t -test). ( b ) EphA2 knockdown was confirmed in human TNBC cell lines via immunoblot analysis. ( c ) MDA-MB-231, HCC1395 and BT549 lines were starved and then serum-stimulated in the presence of BrdU to mark proliferating cells. BrdU-positive nuclei were detected using a FITC-conjugated anti-BrdU antibody (green, arrowheads) and all nuclei were visualized using DAPI counterstain (blue). ( d ) Proliferation (% BrdU + nuclei/total nuclei) was significantly reduced in EphA2 KD lines relative to controls (* P <0.05, Student’s t -test). Boxed areas in upper vector control panels indicate areas of high magnification displayed in lower panels. ( e ) For apoptosis assays, cells were starved for 48 h and then subjected to TUNEL analysis. Apoptosis (% TUNEL + nuclei/total nuclei) was not affected by EphA2 knockdown (not significant, Student’s t -test). N =3 independent experiments with triplicate biological replicates for each line.

Article Snippet: For EphA2 expression analyses, EphA2 protein expression in TNBC cores were compared to expression in normal/hyperplastic and benign fibroadenoma control samples from Cybrdi TMA CC08-10-001.

Techniques: Knockdown, Stable Transfection, Expressing, Control, Plasmid Preparation, shRNA, MTT Assay, Western Blot, TUNEL Assay

Journal: Oncogene

Article Title: Targeting EphA2 impairs cell cycle progression and growth of basal-like/triple-negative breast cancers

doi: 10.1038/onc.2017.170

Figure Lengend Snippet: EphA2 knockdown impairs proliferation in human TNBC lines in vivo . ( a ) MDA-MB-231 and BT549 vector control and EphA2 knockdown (A2KD) lines were orthotopically injected into the mammary fat pads of nude female mice and tumor volume was measured over the course of 2 weeks. EphA2 knockdown tumors were significantly smaller than vector controls at 1 and/or 2 weeks post injection ( N =5 tumors/condition; * P <0.05, ANOVA). ( b , c ) Histologic analyses of tumor sections for Ki67, a marker of proliferation, revealed a significant decrease in proliferation index (%Ki67 + nuclei/total nuclei; * P <0.05, Student’s t -test) for EphA2 −/− tumors relative to controls. ANOVA, analysis of variance.

Article Snippet: For EphA2 expression analyses, EphA2 protein expression in TNBC cores were compared to expression in normal/hyperplastic and benign fibroadenoma control samples from Cybrdi TMA CC08-10-001.

Techniques: Knockdown, In Vivo, Plasmid Preparation, Control, Injection, Marker

Journal: Oncogene

Article Title: Targeting EphA2 impairs cell cycle progression and growth of basal-like/triple-negative breast cancers

doi: 10.1038/onc.2017.170

Figure Lengend Snippet: EphA2-deficiency impairs growth and progression in the C3-TAg transgenic model of basal-like breast cancer in vivo . We crossed C3-TAg mice with EphA2-deficient mice, generating a cohort of C3-TAg wild-type (EphA2 +/+ ), heterozygous (EphA2 +/− ) and EphA2-deficient (EphA2-/-) mice. ( a ) EphA2-deficient mice formed significantly smaller tumors than wild-type or heterozygous littermates. ( b ) Tumor volume was significantly decreased in EphA2-deficient animals relative to littermate controls (N=9 +/+ , 11 +/− and 9 −/− animals; * P <0.05, one-way ANOVA). ( c ) Morphometric analysis of hematoxylin and eosin-stained tumor sections revealed a significant decrease in mitotic figures (green arrowheads, a ) in EphA2 −/− tumors relative to +/+ controls, as well as decreased cellularity ( n =6 independent tumors/genotype; * P <0.05 Student’s t -test). ( d ) Histologic analyses of tumor sections for proliferation marker Ki67 revealed a significant decrease in proliferation index (%Ki67+ nuclei/total nuclei; * P <0.05, Student’s t -test) for EphA2 −/− tumors relative to controls. ( e , f ) Primary tumor cells from EphA2 +/+ and EphA2 −/− C3-TAg tumors were isolated and grown in three-dimensional spheroid cultures on Matrigel for 10–12 days. EphA2 −/− cells formed significantly smaller colonies than EphA2 +/+ cells ( e , f ; * P <0.05, Student’s t -test). Expression of EphA2 in EphA2 −/− cells via adenoviral transduction rescued colony size ( e , f ; * P <0.05, Student’s t -test) relative to adenovirus GFP control (expression confirmed by fluorescence analysis, lower right panel, e ), restoring growth to near wild-type C3-TAg levels. N =3 independent experiments with triplicate biological replicates for each cell line. ( g ) EphA2-deficiency and overexpression of EphA2 following adenoviral transduction were confirmed by immunoblot analysis.

Article Snippet: For EphA2 expression analyses, EphA2 protein expression in TNBC cores were compared to expression in normal/hyperplastic and benign fibroadenoma control samples from Cybrdi TMA CC08-10-001.

Techniques: Transgenic Assay, In Vivo, Staining, Marker, Isolation, Expressing, Transduction, Control, Fluorescence, Over Expression, Western Blot

Journal: Oncogene

Article Title: Targeting EphA2 impairs cell cycle progression and growth of basal-like/triple-negative breast cancers

doi: 10.1038/onc.2017.170

Figure Lengend Snippet: EphA2 loss impairs cell cycle progression through S-phase in TNBC cells. ( a , b ) MDA-MB-231, HCC1395 and BT549 vector control (V) and EphA2 shRNA knockdown (KD) lines were starved then serum-stimulated for 0, 15 or 24 h prior to propidium iodide labeling and FACS analysis for DNA content. ( a ) All three human shEphA2 knockdown lines showed a general decrease in the percentage of cells in S-phase relative to vector controls: MDA-MB-231 at 15 h post serum stimulation, HCC1395 at 0 and 15 h post serum stimulation, and aneuploidy BT549 at 0, 15 and 24 h post serum stimulation. ( b ) Immunoblot analysis of S-phase cell cycle regulators in vector (V) control versus EphA2 shRNA knockdown (KD) human TNBC lines under basal (serum-starved, −) conditions versus stimulation with serum (+) for 15 h; ( c ) and three independent isolates of wild-type (WT) and EphA2-deficient (KO) C3-TAg cells versus primary mouse mammary epithelial cells (MEC). We observed decreased levels of P-Rb, c-Myc and cyclin E2 (green *) and increased expression of p27/KIP1 cell cycle inhibitor (red *) in KD and KO cells relative to controls. Data were from three independent experiments. ( d , e ) Adenovirus-mediated overexpression of EphA2 (Ad.EphA2) significantly increased colony size of wild-type (WT) C3-TAg cells in three-dimensional spheroid culture relative to adenovirus GFP (Ad.GFP) control (* P <0.05, Student’s t -test). Overexpression of adenovirus c-Myc (Ad.c-Myc) in EphA2-deficient (KO) C3-TAg cells rescued three-dimensional spheroid growth to near wild-type levels relative to Ad.GFP controls (* P <0.05, Student’s t -test). ( f ) Immunoblot analyses confirmed EphA2 or c-Myc overexpression as well as modulation of P-Rb, cyclin E2 and p27/KIP1 in WT and KO C3-TAg cells. ( g , h ) Stable knockout of p27/KIP1 by two independent guide RNA sequences also rescued growth of KO C3-TAg cells in spheroid culture. ( i ) Immunoblot analysis confirmed p27/KIP1 knockout. N =three independent experiments/condition with triplicate biological replicates, (* P <0.05, Student’s t -test).

Article Snippet: For EphA2 expression analyses, EphA2 protein expression in TNBC cores were compared to expression in normal/hyperplastic and benign fibroadenoma control samples from Cybrdi TMA CC08-10-001.

Techniques: Plasmid Preparation, Control, shRNA, Knockdown, Labeling, Western Blot, Expressing, Over Expression, Knock-Out

Journal: Oncogene

Article Title: Targeting EphA2 impairs cell cycle progression and growth of basal-like/triple-negative breast cancers

doi: 10.1038/onc.2017.170

Figure Lengend Snippet: Pharmacologic inhibition of EphA2 recapitulates effects of genetic ablation. ( a – c ) MDA-MB-231 and wild-type (WT) C3-TAg tumor cells were grown in the presence of increasing concentrations of an EphA2 small molecule tyrosine kinase inhibitor ALW-II-41-27 (ALW) versus analog control and growth assessed by MTT assay after 72 h in culture. ( a , c ) ALW significantly reduced growth in a dose-dependent manner (* P <0.05, Student’s t -test). ( b , d ) ALW treatment reduced basal levels of P-EphA2/EphA2 ( b , MDA-MB-231 cell line), c-Myc, P-Rb, cyclin E2 and enhanced expression of p27/KIP1 relative to NG-25 control. ( e ) C3-TAg cells were starved and stimulated for 0, 15 and 30 min with ephrin-A1-Fc (EphA2 ligand; 1 μg/ml) in the presence or absence of ALW-II-41-27 or NG-25 control. EphA2 was immunoprecipitated and products probed for tyrosine phosphorylation and EphA2. ALW-II-41-27 significantly reduced basal and ephrin-A1-Fc induced tyrosine phosphorylation. ( f , g ) Wild-type (WT) and EphA2-deficient (KO) C3-TAg cells were grown in three-dimensional spheroid culture in the presence of 1 μ m EphA2 small molecule inhibitor (ALW-II-41-27) versus analog control (NG-25) for 7 days. ( g ) ALW-II-41-27 significantly reduced colony size in WT cells relative to NG-25, but did not affect growth of KO cells, suggesting minimal off-target effects of the compound. N =three independent experiments/condition with triplicate biological replicates, Student’s t -test (NG-25 versus ALW WT cells, P <0.05; NG-25 versus ALW KO cells, not significant; Student’s t -test). ( h – j ) Two independent TNBC patient-derived xenograft (PDX) lines were implanted into the cleared fat pads of female NOD-SCID mice. ( h ) These PDX lines overexpressed EphA2 protein relative to normal/hyperplastic/benign human breast tissue controls. ( i ) Once tumors reached a volume ⩾200 mm 3 , mice were randomized and treated with either vehicle control or ALW-II-41-27 for 7 days. ALW treatment significantly reduced tumor volume over time in both lines (* P <0.05, one-way ANOVA) within 3–5 days after treatment. ( j ) Histologic analyses of tumor sections for proliferation marker revealed a significant decrease in proliferation index (%Ki67 + nuclei/total nuclei; * P <0.05, Student’s t -test) for ALW-treated tumors relative to controls.

Article Snippet: For EphA2 expression analyses, EphA2 protein expression in TNBC cores were compared to expression in normal/hyperplastic and benign fibroadenoma control samples from Cybrdi TMA CC08-10-001.

Techniques: Inhibition, Control, MTT Assay, Expressing, Immunoprecipitation, Phospho-proteomics, Derivative Assay, Marker