Journal: Cancers

Article Title: Drug-Induced Resistance and Phenotypic Switch in Triple-Negative Breast Cancer Can Be Controlled via Resolution and Targeting of Individualized Signaling Signatures

doi: 10.3390/cancers13195009

Figure Lengend Snippet: TNBC tissues are represented by different patient-specific signaling signatures, majority of which do not include EGFR. ( A ) Fold changes in expression levels of EGFR and pEGFR in TNBC and non-TNBC tumors are shown. Y1068 and Y1173 are EGFR phosphorylation sites; ( B ) Examples for patient-specific sets of active unbalanced processes are shown. Each sample harbors a set of 1–3 active unbalanced processes (PaSSS), represented schematically by a barcode. In each barcode active unbalanced processes are represented by black or gray squares, inactive white. Negative/positive amplitude denotes how the patients are correlated with respect to a particular process. Suggested PaSSS-based therapies appear below each barcode; ( C ) Heterogeneity index of 3 subgroups, represented by a ratio between the number of distinct PaSSSs and the number of samples in each subset, is shown for the TNBC subset of tissues, the entire set (3467 samples from 11 cancer types) and the subset of non-TNBC samples. (Abbreviations: TNBC—Triple Negative Breast Cancer, PaSSS—Patient-specific signaling signature, EGFR—Epidermal Growth Factor Receptor, VEGFR2—Vascular Endothelial Growth Factor Receptor 2, Her2—Human Epidermal growth factor Receptor 2, Src—Proto-oncogene tyrosine-protein kinase Src).

Article Snippet: The following conjugated antibodies were used: anti-p-EGFR (Y1068) (R&D Systems, Minneapolis, MN, USA, cat. no. IC3570G), anti-p-ERK2 (Thr202/Tyr204) (BioLegend, San Diego, CA, USA, cat. No. 675503), anti-p-S6 (Ser235/236) (BioLegend, cat. no. 608605), and anti-GAPDH (Santa Cruz Biotechnology, Dallas, Texas, USA, cat. no. sc-47724AF594).

Techniques: Expressing, Phospho-proteomics

Journal: Oncogene

Article Title: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

doi: 10.1038/onc.2012.446

Figure Lengend Snippet: Figure 1. Aki1 expression and association to EGFR in EGFR mutant human lung cancer cell lines. (a) EGFR mutant human lung cancer cell lines (PC-9, HCC827 and H1975), EGFR wild-type human lung cancer cell lines (A549 and PC14PE6) and human lung fibroblast cell lines (MRC-5 and IMR-90) were lysed and the indicated proteins were detected by western blotting. (b) EGFR mutant lung cancer cell lines were treated with or without EGF (50 ng/ml) or IGF-1 (50 ng/ml) for 10 min. Then, cells were lysed and the indicated proteins were detected by western blotting with or without immunoprecipitation of Aki1.

Article Snippet: Oncogene (2013) 4427 – 4435 & 2013 Macmillan Publishers Limited (40D4), anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Par-4, anti-cleaved PARP (Asp214), anti-phospho-S6 ribosomal protein (Ser235/ 236), anti-S6 ribosomal protein (5G10), anti-phospho-IGF-1 R (Tyr1131), DYKDDDDK (FLAG) tag antibody or anti-b-actin (13E5) antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Aki1 (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), anti-PDK1 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human EGFR (1 mg/ml) or anti-human IGF-1 R (0.1mg/ml) antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Mutagenesis, Western Blot, Immunoprecipitation

Journal: Oncogene

Article Title: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

doi: 10.1038/onc.2012.446

Figure Lengend Snippet: Figure 3. Comparison of efficacy between Aki1 knockdown and EGFR inhibition on cell viability. Cells were treated with Aki1-1 siRNA, EGFR siRNA, control scramble siRNA or erlotinib (1 mM). (a) After 72-h incubation, cell viability was determined by MTT assay. (b) After 24-h incubation, cells were lysed and the indicated proteins were detected by western blotting.

Article Snippet: Oncogene (2013) 4427 – 4435 & 2013 Macmillan Publishers Limited (40D4), anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Par-4, anti-cleaved PARP (Asp214), anti-phospho-S6 ribosomal protein (Ser235/ 236), anti-S6 ribosomal protein (5G10), anti-phospho-IGF-1 R (Tyr1131), DYKDDDDK (FLAG) tag antibody or anti-b-actin (13E5) antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Aki1 (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), anti-PDK1 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human EGFR (1 mg/ml) or anti-human IGF-1 R (0.1mg/ml) antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Comparison, Knockdown, Inhibition, Control, Incubation, MTT Assay, Western Blot

Journal: Oncogene

Article Title: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

doi: 10.1038/onc.2012.446

Figure Lengend Snippet: Figure 4. Therapeutic effects of Aki1 knockdown against lung cancer cells with EGFR T790M secondary mutation in vivo. H1975 cells (5 106 cells per 100 ml of PBS) were injected subcutaneously into the flanks of 5-week-old male SCID mice. After cell inoculation, 50 mg of either scramble or Aki1 siRNA complexed with invivofectamine was injected intratumorally on days 5 and 8. (a) Tumor size was measured twice a week and tumor volume was calculated as described in Materials and methods. (b) Macroscopic appearance of the tumors harvested on day 26. (c) The harvested tumors were examined for Aki1, and the inhibition of downstream signaling molecule, S6, in tumors by western blotting.

Article Snippet: Oncogene (2013) 4427 – 4435 & 2013 Macmillan Publishers Limited (40D4), anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Par-4, anti-cleaved PARP (Asp214), anti-phospho-S6 ribosomal protein (Ser235/ 236), anti-S6 ribosomal protein (5G10), anti-phospho-IGF-1 R (Tyr1131), DYKDDDDK (FLAG) tag antibody or anti-b-actin (13E5) antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Aki1 (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), anti-PDK1 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human EGFR (1 mg/ml) or anti-human IGF-1 R (0.1mg/ml) antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Knockdown, Mutagenesis, In Vivo, Injection, Inhibition, Western Blot

Journal: Oncogene

Article Title: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

doi: 10.1038/onc.2012.446

Figure Lengend Snippet: Figure 2. Effects of Aki1 siRNA on cell viability and apoptosis in EGFR mutant human lung cancer cell lines. Cells were treated with Aki1-1 or control scramble siRNA. (a) After 72-h incubation, cell viability was determined by MTT assay. (b) After 24-h incubation with control scramble siRNA (lanes 1, 3 and 5) or Aki1-1 siRNA (lanes 2, 4 and 6), cells were lysed and the indicated proteins were detected by western blotting. (c) After 48-h incubation, cell apoptosis was determined with an Annexin V-FITC Apoptosis Detection Kit I. The numbers show percentages of early apoptotic cells.

Article Snippet: Oncogene (2013) 4427 – 4435 & 2013 Macmillan Publishers Limited (40D4), anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Par-4, anti-cleaved PARP (Asp214), anti-phospho-S6 ribosomal protein (Ser235/ 236), anti-S6 ribosomal protein (5G10), anti-phospho-IGF-1 R (Tyr1131), DYKDDDDK (FLAG) tag antibody or anti-b-actin (13E5) antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Aki1 (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), anti-PDK1 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human EGFR (1 mg/ml) or anti-human IGF-1 R (0.1mg/ml) antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Mutagenesis, Control, Incubation, MTT Assay, Western Blot

Journal: Oncogene

Article Title: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

doi: 10.1038/onc.2012.446

Figure Lengend Snippet: Figure 5. Effects of Aki1 knockdown combined with new generation EGFR TKI in lung cancer with EGFR T790M secondary mutation. H1975 cells were treated with Aki1-1 or control scramble siRNA in the presence or absence of erlotinib (1 mM), CL-387, 785 (0.3 mM), BIBW2992 (0.1 mM) or WZ4002 (0.1 mM). (a) After 72-h incubation, cell viability was determined by MTT assay. *Po0.01, one-way ANOVA. (b) After 24-h incubation, cells were lysed and the indicated proteins were detected by western blotting. (c) Schema showing the role of Aki1 in cells with wild-type EGFR and mutant EGFR.

Article Snippet: Oncogene (2013) 4427 – 4435 & 2013 Macmillan Publishers Limited (40D4), anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Par-4, anti-cleaved PARP (Asp214), anti-phospho-S6 ribosomal protein (Ser235/ 236), anti-S6 ribosomal protein (5G10), anti-phospho-IGF-1 R (Tyr1131), DYKDDDDK (FLAG) tag antibody or anti-b-actin (13E5) antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Aki1 (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), anti-PDK1 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human EGFR (1 mg/ml) or anti-human IGF-1 R (0.1mg/ml) antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Knockdown, Mutagenesis, Control, Incubation, MTT Assay, Western Blot

Journal: Oncogene

Article Title: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

doi: 10.1038/onc.2012.446

Figure Lengend Snippet: Figure 6. Aki1 is frequently expressed in EGFR mutant lung cancer. Clinical specimens from EGFR mutant lung cancer patients were stained for Aki1 by immunohistochemistry. (a) A total of 56 tumor specimens with EGFR-activating mutations were obtained from 56 lung adenocarcinoma patients. Of the 56 patients, 42 were EGFR-TKI naive, 7 tumors were from patients who showed intrinsic resistance to the EGFR-TKIs, gefitinib or erlotinib. Another seven tumors were from patients who showed acquired resistance to EGFR-TKIs. Of 42 EGFR-TKI naive tumors, the presence of Aki1 protein was scored as 2 þ in 31 tumors (74%), 1 þ in 8 tumors (19%) and – in 3 tumors (7%). Aki1 protein was detected diffusely in all of seven tumors with intrinsic resistance: 2 þ in 4 (57%), 1 þ in 3 (43%). Aki1 protein was detected diffusely in all of seven tumors with acquired resistance: 2 þ in 6 (86%), 1 þ in 1 (14%). (b) Representative staining results are shown.

Article Snippet: Oncogene (2013) 4427 – 4435 & 2013 Macmillan Publishers Limited (40D4), anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Par-4, anti-cleaved PARP (Asp214), anti-phospho-S6 ribosomal protein (Ser235/ 236), anti-S6 ribosomal protein (5G10), anti-phospho-IGF-1 R (Tyr1131), DYKDDDDK (FLAG) tag antibody or anti-b-actin (13E5) antibodies (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Aki1 (1:1000 dilution; Bethyl Laboratories, Montgomery, TX, USA), anti-PDK1 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human EGFR (1 mg/ml) or anti-human IGF-1 R (0.1mg/ml) antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Mutagenesis, Staining, Immunohistochemistry