Journal: Oncogene

Article Title: Akt phosphorylates and activates HSF-1 independent of heat shock, leading to Slug overexpression and epithelial-mesenchymal transition (EMT) of HER2-overexpressing breast cancer cells

doi: 10.1038/onc.2013.582

Figure Lengend Snippet: ( a ) Heregulin (HRG) induced EMT of HER2-amplified breast cancer cells. MDA-MB-453 and BT-474 were serum-starved overnight (day 0) and then treated with heregulin (100 ng/ml) for 1 or 3 days. Cultured cells were imaged using a phase-contract microscope. Representative images are shown. ( b ) Slug transcripts were induced by heregulin in HER2-amplified breast cancer cells. MDA-MB-453 and BT-474 were starved from serum overnight, treated with heregulin (100 ng/ml) for 0–120 min, and harvested for total RNA extraction and RT-qPCR. Levels of EMT regulators, Slug, Snail and TWIST were determined. * indicates p-values < 0.05. ( c ) ( d ) Slug protein expression was induced by heregulin in HER2-amplified breast cancer cells. MDA-MB-453 and BT-474 were starved from serum overnight, treated with heregulin (100 ng/ml) for 0–120 min ( c ) or for 1–3 days ( d ), and harvested for protein extraction and WB. Levels of Slug, E-cadherin (epithelial marker), and Vimentin (mesenchymal marker) were analyzed. ( e ) Slug promoter was significantly activated by heregulin in breast cancer cells with HER2 amplification. MDA-MB-453, BT-474 and SK-BR-3 cells were transfected with a firefly luciferase reporter under the control of the human Slug promoter, serum-starved for 16 hrs and treated with heregulin (100 ng/ml) for 2 hrs. Treated cells were lysed and subjected to luciferase assay. All cells were co-transfected with the Renilla luciferase expression vector, pRL-CMV, to control for transfection efficiency. The results were derived from at least three experiments. The student t-test was conducted to compute p-values. * indicates p-values < 0.05.

Article Snippet: This was performed as described previously., Antibodies used in WB included mouse monoclonal antibodies against β-actin (Sigma), α-tubulin (Sigma), E-cadherin (610404, BD), p-HSF-1/S326 (ab76076, Epitomics/Abcam), p-JNK/S63/73 (sc-6254; Santa Cruz), as well as, rabbit antibodies against Slug (AP2053a,ABGENT), HSF-1 (4356, Cell Signaling), Hsp70 (4876, Cell Signaling), p38 (9212, Cell Signaling), p-p38/T180/Y182 (4511, Cell Signaling), pERK/T202/Y204 (9109, Cell Signaling), Akt/pan (4691, Cell Signaling), p-Akt/S473 (4060, Cell Signaling), mTOR (2983, Cell Signaling), p-mTOR/S2448 (5536, Cell Signaling), JNK (sc-571, Santa Cruz), HER2 (2165, Cell Signaling), p-HER2/Y1278 (2247, Cell Signaling).

Techniques: Amplification, Cell Culture, Microscopy, RNA Extraction, Quantitative RT-PCR, Expressing, Protein Extraction, Marker, Transfection, Luciferase, Control, Plasmid Preparation, Derivative Assay

Journal: Oncogene

Article Title: Akt phosphorylates and activates HSF-1 independent of heat shock, leading to Slug overexpression and epithelial-mesenchymal transition (EMT) of HER2-overexpressing breast cancer cells

doi: 10.1038/onc.2013.582

Figure Lengend Snippet: ( a )( b ) Ectopic HER2 expression induced Slug expression in breast cancer cells. MCF-7 (with normal HER2 expression level) and MCF-7/HER2 (MCF-7 cells stably expressing ectopic HER2) were analyzed for Slug and E-cadherin expression using RT-PCR ( a ) and WB ( b ). Enhanced HER2 expression in MCF-7/HER2 cells is indicated by WB. ( c )( d ) Ectopic HER2 expression induced EMT-like morphology changes in breast cancer cells. In panel c, both cell lines were cultured in normal growth condition with fetal calf serum. In panel d, cells were serum-starved and treated with heregulin (100 ng/ml) for 0–3 days. Representative images are shown. ( e ) Ectopic HER2 expression induced Slug promoter activity in breast cancer cells. MCF-7 cells (with normal HER2 levels) were transiently transfected with HER2 and the Slug luciferase reporter, serum-starved, and then stimulated with heregulin (100 ng/ml) for 2 hrs. All cells were co-transfected with the Renilla luciferase expression vector, pRL-CMV, to control for transfection efficiency. The results were derived from at least three experiments, and analyzed by the student t-test to compute p-values. * indicates p-values < 0.05. ( f ) Lapatinib, a small molecule HER2/EGFR inhibitor reduced Slug expression in HER2-amplified MDA-MB-453 and SK-BR-3 cells. Cells were pre-treated with lapatinib (5 uM) for 24 hrs and subjected to total RNA extraction and RT-PCR for Slug transcript levels.

Article Snippet: This was performed as described previously., Antibodies used in WB included mouse monoclonal antibodies against β-actin (Sigma), α-tubulin (Sigma), E-cadherin (610404, BD), p-HSF-1/S326 (ab76076, Epitomics/Abcam), p-JNK/S63/73 (sc-6254; Santa Cruz), as well as, rabbit antibodies against Slug (AP2053a,ABGENT), HSF-1 (4356, Cell Signaling), Hsp70 (4876, Cell Signaling), p38 (9212, Cell Signaling), p-p38/T180/Y182 (4511, Cell Signaling), pERK/T202/Y204 (9109, Cell Signaling), Akt/pan (4691, Cell Signaling), p-Akt/S473 (4060, Cell Signaling), mTOR (2983, Cell Signaling), p-mTOR/S2448 (5536, Cell Signaling), JNK (sc-571, Santa Cruz), HER2 (2165, Cell Signaling), p-HER2/Y1278 (2247, Cell Signaling).

Techniques: Expressing, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Control, Derivative Assay, Amplification, RNA Extraction

Journal: Oncogene

Article Title: Akt phosphorylates and activates HSF-1 independent of heat shock, leading to Slug overexpression and epithelial-mesenchymal transition (EMT) of HER2-overexpressing breast cancer cells

doi: 10.1038/onc.2013.582

Figure Lengend Snippet: ( a ) Heregulin induced phosphorylation of both HSF-1 and Akt in HER2-amplified breast cancer cell lines. MDA-MB-453 and BT-474 cells were treated with and without heregulin (100 ng/ml) for 2 hrs and the whole cell lysates were analyzed by WB for levels of HER2 downstream kinases and Slug. ( b ) Kinetics for HSF-1 activation is in concordance with that for Akt. The two cell lines were treated with heregulin for 0–240 min and the whole cell lysates were analyzed by WB to determine levels of p-HSF-1 (S326) and p-Akt (S473). ( c ) Ectopic HER2 expression led to increased activation of both HSF-1 and Akt. MCF-7 and MCF-7/HER2 cell lines were examined using WB.

Article Snippet: This was performed as described previously., Antibodies used in WB included mouse monoclonal antibodies against β-actin (Sigma), α-tubulin (Sigma), E-cadherin (610404, BD), p-HSF-1/S326 (ab76076, Epitomics/Abcam), p-JNK/S63/73 (sc-6254; Santa Cruz), as well as, rabbit antibodies against Slug (AP2053a,ABGENT), HSF-1 (4356, Cell Signaling), Hsp70 (4876, Cell Signaling), p38 (9212, Cell Signaling), p-p38/T180/Y182 (4511, Cell Signaling), pERK/T202/Y204 (9109, Cell Signaling), Akt/pan (4691, Cell Signaling), p-Akt/S473 (4060, Cell Signaling), mTOR (2983, Cell Signaling), p-mTOR/S2448 (5536, Cell Signaling), JNK (sc-571, Santa Cruz), HER2 (2165, Cell Signaling), p-HER2/Y1278 (2247, Cell Signaling).

Techniques: Phospho-proteomics, Amplification, Activation Assay, Expressing

Journal: Oncogene

Article Title: Akt phosphorylates and activates HSF-1 independent of heat shock, leading to Slug overexpression and epithelial-mesenchymal transition (EMT) of HER2-overexpressing breast cancer cells

doi: 10.1038/onc.2013.582

Figure Lengend Snippet: In luciferase assays (panels b, d and e), three independent experiments were performed to derive means and standard deviations. All cells were co-transfected with the Renilla luciferase reporter, pRL-CMV, to control for transfection efficiency. The results were analyzed by the student t-test to compute p-values. * indicates p-values < 0.05. ( a )( b ) Small molecule inhibitors to PI3K/Akt (LY294002; LY; 50 uM) and HER2 (Lapatinib; Lap; 25 uM) pre-treatment for 1 2 hrs suppressed heregulin-induced Slug expression in BT-474 cells. In panel a, WB was conducted. Hsp70 served as positive controls for HSF-1 activity. Heregulin exposure was for 1 hr at 100 ng/ml. In panel b, total RNA was analyzed by RT-PCR. Veh, vehicle (1% DMSO) ( c ) Both LY294002 and Lapatinib blocked heregulin induction of Slug promoter activity. BT-474 cells transfected with the Slug luciferase reporter were serum-starved, treated with vehicle or indicated inhibitor (50 uM LY294002 or 25 uM Lapatinib) for 2 hrs, and then treated with and without heregulin for 4 hrs. Harvested cells were lysed and subjected to luciferase assay. ( d ) HSF-1 and Akt siRNAs reduced Slug expression in BT-474 cells, as shown by WB. ( e ) HSF-1 and Akt siRNAs prevented heregulin-induced activation of Slug promoter in BT-474 cells. BT-474 cells co-transfected with siRNA and the Slug luciferase reporter were serum-starved and treated with heregulin for 4 hrs, and then subjected to luciferase assay. ( f ) DN-Akt blocked heregulin-induced Slug expression and HSF-1 activation in MCF-7/HER2 cells, as shown by WB. ( g ) Akt siRNA and DN-Akt inhibited Slug transcription in BT-474 cells as shown by RT-PCR. ( h ) DN-Akt reduced activity of the Slug promoter in MCF-7/HER2 cells while CA-Akt enhanced its activity in MCF-7 cells. Luciferase assay was conducted to measure Slug promoter activity. ( i ) HSF-1 is essential for CA-Akt-induced Slug expression. MCF-7 cells were used. Left, luciferase assay. Right, WB.

Article Snippet: This was performed as described previously., Antibodies used in WB included mouse monoclonal antibodies against β-actin (Sigma), α-tubulin (Sigma), E-cadherin (610404, BD), p-HSF-1/S326 (ab76076, Epitomics/Abcam), p-JNK/S63/73 (sc-6254; Santa Cruz), as well as, rabbit antibodies against Slug (AP2053a,ABGENT), HSF-1 (4356, Cell Signaling), Hsp70 (4876, Cell Signaling), p38 (9212, Cell Signaling), p-p38/T180/Y182 (4511, Cell Signaling), pERK/T202/Y204 (9109, Cell Signaling), Akt/pan (4691, Cell Signaling), p-Akt/S473 (4060, Cell Signaling), mTOR (2983, Cell Signaling), p-mTOR/S2448 (5536, Cell Signaling), JNK (sc-571, Santa Cruz), HER2 (2165, Cell Signaling), p-HER2/Y1278 (2247, Cell Signaling).

Techniques: Luciferase, Transfection, Control, Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Activation Assay

Journal: Oncogene

Article Title: Akt phosphorylates and activates HSF-1 independent of heat shock, leading to Slug overexpression and epithelial-mesenchymal transition (EMT) of HER2-overexpressing breast cancer cells

doi: 10.1038/onc.2013.582

Figure Lengend Snippet: ( a ) Identification of four putative HSEs within the human Slug gene promoter. TFSearch, a web-based search engine for transcription factor-binding sites, was used to search for HSEs within the Slug promoter. Consensus HSEs are shown on the top. Structures of the wild-type Slug promoter reporter and two mutant reporters are shown. Each of the two mutant promoters contains mutations at two of the four putative sites, in order to destroy the three repeats required for binding to HSF-1 trimers. Clear boxes mark the putative HSEs. Lower case letters indicate mutated bases. ( b ) HSF-1 binds to the Slug promoter and the binding was enhanced by heregulin. Serum-starved BT-474 cells treated with and without heregulin (100 ng/ml) were used in the ChIP assay. HSF-1 antibody (Ab) was used to immunoprecipitate HSF-1 while IgG served as the negative controls. Chromatin input was used to control for loading. PCR was conducted to detect HSF-1 binding to Slug promoter and a known HSF-1 target gene, Hsp70. ( c ) Ectopic HSF-1 expression significantly induced Slug promoter activity. Cells were transfected with the control vector or the HSF-1 vector, and the Slug luciferase reporter for 48 hrs and subjected to luciferase assay. All cells were co-transfected with the Renilla luciferase expression vector, pRL-CMV, to control for transfection efficiency. The results represent means and standard deviations from at least three experiments, and were analyzed by the student t-test to compute p-values. * indicates p-values < 0.05. ( d ) Ectopic HSF-1 expression enhanced Slug expression. BT-474 and MDA-MB-453 cells transfected with the control vector or the HSF-1 vector were analyzed by WB to determine Slug and HSF-1 expression levels. ( e )( f ) Identified HSEs are important for heregulin- and HSF-1-mediated induction of Slug promoter activation. MDA-MB-453 cells transfected with the wild-type and mutant slug reporters were serum-starved and treated with heregulin for 2 hrs, and then subjected to luciferase assay. All cells were co-transfected with the Renilla luciferase reporter, pRL-CMV, to control for transfection efficiency. The results were derived from at least three experiments, and analyzed by the student t-test to compute p-values. * indicates p-values < 0.05. ( g ) Levels of p-HSF-1 (S326) were directly associated with those of Slug in invasive breast carcinoma specimens. IHC was conducted to analyze 100 invasive carcinomas. Linear regression was used to compute R and p values (R=0.56, p<0.000001). Right, representative images.

Article Snippet: This was performed as described previously., Antibodies used in WB included mouse monoclonal antibodies against β-actin (Sigma), α-tubulin (Sigma), E-cadherin (610404, BD), p-HSF-1/S326 (ab76076, Epitomics/Abcam), p-JNK/S63/73 (sc-6254; Santa Cruz), as well as, rabbit antibodies against Slug (AP2053a,ABGENT), HSF-1 (4356, Cell Signaling), Hsp70 (4876, Cell Signaling), p38 (9212, Cell Signaling), p-p38/T180/Y182 (4511, Cell Signaling), pERK/T202/Y204 (9109, Cell Signaling), Akt/pan (4691, Cell Signaling), p-Akt/S473 (4060, Cell Signaling), mTOR (2983, Cell Signaling), p-mTOR/S2448 (5536, Cell Signaling), JNK (sc-571, Santa Cruz), HER2 (2165, Cell Signaling), p-HER2/Y1278 (2247, Cell Signaling).

Techniques: Binding Assay, Mutagenesis, Control, Expressing, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Activation Assay, Derivative Assay

Journal: Oncogene

Article Title: Akt phosphorylates and activates HSF-1 independent of heat shock, leading to Slug overexpression and epithelial-mesenchymal transition (EMT) of HER2-overexpressing breast cancer cells

doi: 10.1038/onc.2013.582

Figure Lengend Snippet: In luciferase assays (panels b, d and e), three independent experiments were performed to derive means and standard deviations. All cells were co-transfected with the Renilla luciferase reporter, pRL-CMV, to control for transfection efficiency. The results were analyzed by the student t-test to compute p-values. * indicates p-values < 0.05. ( a )( b ) Small molecule inhibitors to PI3K/Akt (LY294002; LY; 50 uM) and HER2 (Lapatinib; Lap; 25 uM) pre-treatment for 1 2 hrs suppressed heregulin-induced Slug expression in BT-474 cells. In panel a, WB was conducted. Hsp70 served as positive controls for HSF-1 activity. Heregulin exposure was for 1 hr at 100 ng/ml. In panel b, total RNA was analyzed by RT-PCR. Veh, vehicle (1% DMSO) ( c ) Both LY294002 and Lapatinib blocked heregulin induction of Slug promoter activity. BT-474 cells transfected with the Slug luciferase reporter were serum-starved, treated with vehicle or indicated inhibitor (50 uM LY294002 or 25 uM Lapatinib) for 2 hrs, and then treated with and without heregulin for 4 hrs. Harvested cells were lysed and subjected to luciferase assay. ( d ) HSF-1 and Akt siRNAs reduced Slug expression in BT-474 cells, as shown by WB. ( e ) HSF-1 and Akt siRNAs prevented heregulin-induced activation of Slug promoter in BT-474 cells. BT-474 cells co-transfected with siRNA and the Slug luciferase reporter were serum-starved and treated with heregulin for 4 hrs, and then subjected to luciferase assay. ( f ) DN-Akt blocked heregulin-induced Slug expression and HSF-1 activation in MCF-7/HER2 cells, as shown by WB. ( g ) Akt siRNA and DN-Akt inhibited Slug transcription in BT-474 cells as shown by RT-PCR. ( h ) DN-Akt reduced activity of the Slug promoter in MCF-7/HER2 cells while CA-Akt enhanced its activity in MCF-7 cells. Luciferase assay was conducted to measure Slug promoter activity. ( i ) HSF-1 is essential for CA-Akt-induced Slug expression. MCF-7 cells were used. Left, luciferase assay. Right, WB.

Article Snippet: This was performed as described previously., Antibodies used in WB included mouse monoclonal antibodies against β-actin (Sigma), α-tubulin (Sigma), E-cadherin (610404, BD), p-HSF-1/S326 (ab76076, Epitomics/Abcam), p-JNK/S63/73 (sc-6254; Santa Cruz), as well as, rabbit antibodies against Slug (AP2053a,ABGENT), HSF-1 (4356, Cell Signaling), Hsp70 (4876, Cell Signaling), p38 (9212, Cell Signaling), p-p38/T180/Y182 (4511, Cell Signaling), pERK/T202/Y204 (9109, Cell Signaling), Akt/pan (4691, Cell Signaling), p-Akt/S473 (4060, Cell Signaling), mTOR (2983, Cell Signaling), p-mTOR/S2448 (5536, Cell Signaling), JNK (sc-571, Santa Cruz), HER2 (2165, Cell Signaling), p-HER2/Y1278 (2247, Cell Signaling).

Techniques: Luciferase, Transfection, Control, Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Activation Assay