Journal: Oncogene

Article Title: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin.

doi: 10.1038/onc.2010.180

Figure Lengend Snippet: Figure 1 Elevated expression of erbB3 via stable transfection induces Akt activation and attenuates paclitaxel cyototoxicity in erbB2-overexpressing breast cancer cells. (a) MDA-MB-231 and SKBR3 cells were transfected with either pDsRed-Monimer-N1 or pDsRed-erbB3. After a 2-month selection with Geneticin, the stable clones of vector control (neo.1) or human erbB3 compli- mentary DNA (cDNA; B3.pool, B3.1, B3.2) were established, and the total cell lysates were subjected to western blot analyses with specific antibodies directed against erbB3, erbB2, epidermal growth factor receptor (EGFR), P-Akt, Akt, P- mitogen-activated protein kinase (P-MAPK), MAPK or b-actin. (b, c) The series of stable transfectants derived from MDA-MB-231 or SKBR3 cells were plated onto 96-well plates with complete culture medium (Dulbecco’s modified Eagle medium (DMEM)/F12, 10% fetal bovine serum (FBS)). After 24 h, the medium was replaced with control medium (fresh DMEM/F12, 0.5% FBS) or same medium containing indicated concentrations of paclitaxel for another 72-h incubation. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy- methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)). Data shows the representative of at least three independent experiments. Bars represent s.d. values.

Article Snippet: Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC75) (from Sigma).

Techniques: Expressing, Stable Transfection, Activation Assay, Transfection, Selection, Clone Assay, Plasmid Preparation, Control, Western Blot, Derivative Assay, Incubation

Journal: Oncogene

Article Title: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin.

doi: 10.1038/onc.2010.180

Figure Lengend Snippet: Figure 2 Transient induction of erbB3 expression activates Akt and inhibits paclitaxel-induced apoptosis in erbB2-overexpressing breast cancer cells. MDA-MB-231 (MDA-231) and SKBR3 cells were infected with lentivirus containing either control vector or pLEX-erbB3 for 24 h. After selection with puromycin (1 mg/ml) for 48 h, the cells were subjected to the following experiments. (a) Western blot analyses of erbB3, P-erbB3, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK or b-actin. (b) MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)) assays to determine the percentages of surviving cells upon paclitaxel treatment from each cell line relative to controls, defined as 100% survival. Data shows the representative of at least three independent experiments. Bars represent s.d. values. (c, d) The cells untreated or treated with paclitaxel (5 nM for SKBR3 cells, 20 nM for MDA-MB-231 cells), for additional 24 h, were then collected and subjected to western blot analyses of poly (ADP-ribose) polymerase (PARP; F-PARP, full length PARP; C-PARP, cleaved PARP), caspase-3 (F-Casp3, full-length caspase-3; C-Casp3, cleaved caspase-3) or b-actin (c) and apoptosis enzyme-linked immunosorbent assay (ELISA) (d).

Article Snippet: Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC75) (from Sigma).

Techniques: Expressing, Infection, Control, Plasmid Preparation, Selection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin.

doi: 10.1038/onc.2010.180

Figure Lengend Snippet: Figure 3 Specific knock down of erbB3 expression reduces P-Akt levels and significantly enhances paclitaxel-induced apoptosis. SKBR3 and MDA-MB-453 (MDA-453) cells were infected with lentivirus containing either ConshRNA or erbB3shRNA for 24 h. After selection with puromycin (1 mg/ml) for 48 h, the cells were subjected to the following experiments. (a) Western blot analyses of erbB3, P-erbB3, erbB2, P-erbB2, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK or b-actin. (b) MTS ((3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)) assays to determine the percentages of surviving cells upon paclitaxel treatment from each cell line relative to controls, defined as 100% survival. Data shows the representative of at least three independent experiments. Bars represent s.d. values. (c, d) The cells untreated or treated with paclitaxel (5 nM) for additional 24 h were collected and subjected to western blot analyses of poly (ADP-ribose) polymerase (PARP), caspase-8 (F-Casp8, full-length caspase-8; C-Casp8, cleaved caspase-8), caspase-3 or b-actin (c) and apoptosis enzyme-linked immunosorbent assay (ELISA) (d).

Article Snippet: Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC75) (from Sigma).

Techniques: Knockdown, Expressing, Infection, Selection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin.

doi: 10.1038/onc.2010.180

Figure Lengend Snippet: Figure 4 The expression levels of erbB3 modulate Survivin expression in erbB2-overexpressing breast cancer cells through a PI-3K/ Akt-dependent mechanism. (a) MDA-MB-231, SKBR3 cells, their stable transfectants, and the MDA-MB-231 and SKBR3 cells infected with lentivirus containing either control vector or pLEX-erbB3, as performed in Figure 2, were collected and subjected to western blot analyses of Survivin, C-X-C chemokine receptor type 4 (CXCR4), Mcl-1, Bcl-xL or b-actin. (b) SKBR3, MDA-MB-453 and 435.eB1 cells infected with lentivirus containing either ConshRNA or erbB3shRNA, as performed in Figure 3, were collected and subjected to western blot analyses of Survivin, CXCR4, Mcl-1, Bcl-xL or b-actin. (c and d) SKBR3.B3.1 and SKBR3.B3.2 cells, untreated or treated with PD98059 (PD, 20 mM) or LY294002 (LY, 10 mM) for 6 h, were then collected and subjected to (c) western blot analyses of Survivin, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK or b-actin and (d) MTS ((3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)) assays to determine the percentages of surviving cells on paclitaxel treatment from each cell line relative to controls, defined as 100% survival. Data shows the representative of at least three independent experiments. Bars represent s.d. values. The densitometry analyses of Survivin signals were shown underneath, and the arbitrary numbers indicate the intensities of each cell line relative to controls, defined as 1.0.

Article Snippet: Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC75) (from Sigma).

Techniques: Expressing, Infection, Control, Plasmid Preparation, Western Blot

Journal: Oncogene

Article Title: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin.

doi: 10.1038/onc.2010.180

Figure Lengend Snippet: Figure 5 Specific knockdown of Survivin expression abrogates erbB3-mediated paclitaxel resistance in erbB2-overexpressing breast cancer cells. (a) SKBR3.B3.1 and SKBR3.B3.2 cells infected with lentivirus containing either ConshRNA or SurshRNA (S3, S4 and S5) were subjected to the following experiments. (a) Western blot analyses of Survivin, erbB2, erbB3, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK or b-actin. (b) MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium,inner salt)) assays to determine the percentages of surviving cells on paclitaxel treatment from each cell line relative to controls, defined as 100% survival. Data shows the representative of at least three independent experiments. Bars represent s.d. values. (c, d) The cells untreated or treated with paclitaxel (5 nM) for additional 24 h were collected and subjected to (c) western blot analyses of poly (ADP-ribose) polymerase (PARP), caspase-8, caspase-3 or b-actin and (d) apoptosis enzyme-linked immunosorbent assay (ELISA). *Po0.005, **P ¼ 0.0133, ***P ¼ 0.0064 vs ConshRNA þ paclitaxel.

Article Snippet: Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC75) (from Sigma).

Techniques: Knockdown, Expressing, Infection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin.

doi: 10.1038/onc.2010.180

Figure Lengend Snippet: Figure 6 Expression of erbB3 forms dimerization with erbB2, but not epidermal growth factor receptor (EGFR), and enhances erbB2 tyrosine kinase activation in breast cancer cells. (a) SKBR3 and MDA-231 cells infected with lentivirus containing either control vector or pLEX-erbB3 along with 435.eB1 and MDA-453 cells were collected and subjected to immunoprecipitation with anti- erbB3 mAb (mouse IgG was used as negative control with the same amount of total lysates of 435.eB1 and MDA-453 cells), and then followed by western blot analyses of erbB2, erbB3 or EGFR. (b) SKBR3 and MDA-231 cells infected with lentivirus containing either control vector or pLEX-erbB3 along with 435.eB1 and MDA-453 cells were collected and subjected to western blot analyses of erbB2, P-erbB2, erbB3, P-erbB3, EGFR, P-EGFR or b-actin. (c) Diagram of proposed model underlying the mechanism of erbB3-mediated paclitaxel resistance in erbB2-overexpressing breast cancer cells.

Article Snippet: Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC75) (from Sigma).

Techniques: Expressing, Activation Assay, Infection, Control, Plasmid Preparation, Immunoprecipitation, Negative Control, Western Blot