Journal: JNCI Journal of the National Cancer Institute

Article Title: Co-activation of STAT3 and YES-Associated Protein 1 (YAP1) Pathway in EGFR-Mutant NSCLC

doi: 10.1093/jnci/djx014

Figure Lengend Snippet: Effects of the triple combination of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), TPCA-1, and AZD0530 in PC-9 and H1975 cells. Extracts from (A) the PC-9 cell line treated with gefitinib (0.05 μM), TPCA-1 (5 μM), or AZD0530 (0.5 μM), or double and triple combinations for 24 hours and (B) the H1975 cell line treated with AZD9291 (0.05 μM), TPCA-1 (5 μM), or AZD0530 (0.5 μM), or double and triple combinations for 24 hours, were analyzed for indicated antibodies as well as β-actin as a loading control to confirm equal gel loading by immunoblot analysis. Similar results were obtained in three independent experiments. C) PC-9 cells were treated with serial dilutions of gefitinib, TPCA-1, AZD0530 alone and with their double and triple combinations for 72 hours. The cell viability was measured by MTT, and the synergy between the drugs was determined using the Chou and Talalay method (Chou and Talalay plot or Fa plot). The dotted horizontal line at 1 indicates the line of additive effect. Effect ( Fa ) indicates the fractional inhibition for each combination index. To calculate drug concentration for each Fa point, the drugs were mixed using constant ratios corresponding to 1/8, 1/4, 1/2, 5/8, 3/4, 7/8, 1, 1.5, and 2 of the individual IC50 values for each drug in the PC-9 cell line. The results represent the means of at least three independent experiments. Data are presented as the means ± standard deviation. D) H1975 cells were treated with serial dilutions of AZD9291, TPCA-1, AZD0530 as a triple combination for 72 hours, a procedure similar to those described in (C) . E) PC-9 cells grown in six-well plates (1000 cells/well) for 24 hours and then left untreated or treated with gefitinib, TPCA-1, and AZD0530 alone and with their double and triple combinations. After 72 hours, media was replaced with fresh media without drugs. After seven more days, cells were washed and stained with crystal violet and then photographed. The crystal violet was extracted and assayed by spectrophotometry. The absorbance was measured at 570 nm. Gefitinib was used at 0.05 μM, TPCA-1 at 5 μM, and AZD0530 at 0.5 μM. Data are means ± standard deviation of three independent experiments. * P = .04, gefitinib plus TPCA-1 vs control; † P = .003, gefitinib plus AZD0530 vs control; ‡ P = .03, gefitinib plus TPCA-1 plus AZD0530 vs gefitinib plus TPCA-1; § P = .05, gefitinib plus TPCA-1 plus AZD0530 vs gefitinib plus AZD0530 (two-sided Student’s t test). BIM = Bcl2 interacting mediator of cell death; EGFR = epidermal growth factor receptor; STAT3 = signal transducer and activator of transcription 3; YAP1 = YES-associated protein 1.

Article Snippet: AZD0530 (SFK inhibitor; saracatinib) and the third-generation EGFR TKI AZD9291 were purchased from Selleck Chemicals.

Techniques: Western Blot, Inhibition, Concentration Assay, Standard Deviation, Staining, Spectrophotometry

Journal: JNCI Journal of the National Cancer Institute

Article Title: Co-activation of STAT3 and YES-Associated Protein 1 (YAP1) Pathway in EGFR-Mutant NSCLC

doi: 10.1093/jnci/djx014

Figure Lengend Snippet: Effect of epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), and Src co-inhibition in vivo. A) Mice with established PC-9 tumors were treated with vehicle control, gefitinib alone, TPCA-1 alone, AZD0530 alone, gefitinib and TPCA-1, gefitinib and AZD0530 or gefitinib, TPCA-1, and AZD0530. Each point represents the mean ± standard deviation of the tumor volume (n = 4 per group). Initially, different concentrations of gefitinib (2, 10, 50 mg/kg) were used. Both the 10 mg/kg and 50 mg/kg dose groups showed tumor regression with complete disappearance after 10 days of treatment. Considering the high sensitivity of PC-9 cells to gefitinib, a very low concentration of gefitinib (2 mg/kg) was finally used in this experiment, which explains the shape of the survival curves. Statistically significant differences on day 30 are shown for gefitinib plus AZD0530 vs gefitinib alone (* P < .001), gefitinib plus TPCA-1 vs gefitinib alone († P < .001), and the triple combination of gefitinib, TPCA-1, and AZD0530 vs gefitinib plus AZD0530 (‡ P < .001) and vs gefitinib plus TPCA-1 (§ P = .01). B) After 30 days, the mice were killed and the tumors removed and weighed. Tumor weights were individually plotted, and comparisons between control and treatment groups were analyzed by Student’s t test. Representative tumor pictures were taken. The reduction in tumor weight obtained with gefitinib plus TPCA-1 or gefitinib plus AZD0530 in the PC-9 xenograft model was statistically significantly different compared with gefitinib alone († P < .001 and * P = .009, respectively). The reduction in tumor weight obtained with the triple combination of gefitinib, TPCA-1, and AZD0530 was statistically significantly different compared with gefitinib plus TPCA-1 (§ P = .03) and gefitinib plus AZD0530 (‡ P < .001). C) Representative tumors surgically removed. The two-sided Student’s t test was used for the statistical analysis. EGFR = epidermal growth factor receptor; STAT3 = signal transducer and activator of transcription 3; YAP1 = YES-associated protein 1.

Article Snippet: AZD0530 (SFK inhibitor; saracatinib) and the third-generation EGFR TKI AZD9291 were purchased from Selleck Chemicals.

Techniques: Inhibition, In Vivo, Standard Deviation, Concentration Assay

Journal: JNCI Journal of the National Cancer Institute

Article Title: Co-activation of STAT3 and YES-Associated Protein 1 (YAP1) Pathway in EGFR-Mutant NSCLC

doi: 10.1093/jnci/djx014

Figure Lengend Snippet: Epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), and Src-YES-associated protein 1 (YAP1) crosstalk. EGFR-activating mutations located in the tyrosine kinase domains and mainly in the form of a base-pair deletion at exon 19 (ΔE746_A750) or a point mutation at exon 21 (L858R) enhance cell growth and invasion via tyrosine phosphorylation and lead to the activation of mitogen-activated protein kinase (MAPK), STAT3, and AKT pathways. Ras-ERK signaling promotes cell growth and decreases apoptosis-related BIM expression. SHP2 modulates signals of receptor tyrosine kinases at the level of Ras. Phosphorylation of the tyrosine residue 705 of cytoplasmic STAT3 in response to activated EGFR promotes STAT3 homodimerization, which leads to nucleus translocation and DNA binding. IL-6 signals via receptor complexes, which contain gp130, the common signal-transducing protein of the IL-6 family of cytokines and IL-6R. IL-6R is not a signal transducer, but its function is to present IL-6 to the signal-transducer gp130, resulting in phosphorylation of gp130 by JAK2 and recruitment of STAT3. In EGFR TKI–resistant cells, paracrine or autocrine stimulation of the TGF-β axis drives expression of IL-6 and activation of STAT3, unleashing the cells from their EGFR activity dependency. gp130 associates with Src and YES and triggers activation of YAP1 through phosphorylation on the tyrosine residue 357, independently of STAT3. YAP1 is normally kept inactive in the cytoplasm through phosphorylation on serine residue 127 by the Hippo effector kinase LATS. EGFR inhibition promotes immediate ubiquitination of TRAF2, which is essential for RIP1 and IKK activation, IkB phosphorylation, and degradation and NF-κB (RelA) nuclear translocation. NF-κB-induced IL-6 ensures STAT3 activation. TPCA-1 is a STAT3 inhibitor. AZD0530 (saracatinib) is a potent, orally administered small molecule that inhibits Src by blocking the ATP-binding site of Src kinases. BIM = Bcl2 interacting mediator of cell death; CTGF = connective tissue growth factor; ERK = extracellular signal-regulated kinase; gp130 = glycoprotein 130; IkB = nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor; IKK = IkB kinase; IL-6 = interleukin 6; IL-6R = IL-6 receptor; JAK2 = Janus kinase 2; LATS1/2 = large tumor suppressor kinase 1 and 2; MEK = mitogen-activated protein kinase; MTOR = mechanistic target of rapamycin; P 70 S6K = ribosomal protein S6 kinase beta-1; NF-κB = nuclear factor kappa light chain enhancer of activated B cells; PI3K = phosphatidylinositide 3 kinase; P XN = paxillin; RelA = v-rel reticuloendotheliosis viral oncogene homolog A; RIP1 = receptor-interacting protein 1; Ser = serine; SHP2 = Src homology region 2–containing protein tyrosine phosphatase 2; STAT3 = signal transducer and activator of transcription 3; TEAD1 = TEA domain transcription factor 1; TGF-beta = transforming growth factor beta; Thr = threonine; TRAF2 = TNF receptor–associated factor 2; Tyr = tyrosine; YAP1 = YES-associated protein 1.

Article Snippet: AZD0530 (SFK inhibitor; saracatinib) and the third-generation EGFR TKI AZD9291 were purchased from Selleck Chemicals.

Techniques: Mutagenesis, Activation Assay, Expressing, Translocation Assay, Binding Assay, Activity Assay, Inhibition, Blocking Assay

Journal: Frontiers in Microbiology

Article Title: Candida albicans : The Ability to Invade Epithelial Cells and Survive under Oxidative Stress Is Unlinked to Hyphal Length

doi: 10.3389/fmicb.2017.01235

Figure Lengend Snippet: C. albicans SC5314 strain induced host cell biphasic ERK 1/2 activation and cortactin phosphorylation by SFKs. HeLa cells were incubated with blastospores from the SC5314 strain for the indicated time points. After incubation, HeLa cells were harvested, lysed, and their protein lysates separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot using the indicated antibodies. The SC5314 strain induced (A) a biphasic activation of host ERK 1/2 (the other isolates did not, not shown) and also (B) the phosphorylation of cortactin (pCort [Y466]) by SFKs. These are representative observations from at least three independent experiments. SC5314, 997,5 g, oral L3837 and blood L3881 isolates induced host cell signaling activation. HeLa cells were incubated with blastospores from four C. albicans isolates for the indicated time points. After incubation, HeLa cells were harvested, lysed, and SFK and cortactin activation were analyzed by Western blot using the indicated antibodies. Cortactin, SFKs and actin have different molecular weights (80–85, 60, and 45 kDa, respectively), thus the nitrocellulose membrane was cut into three horizontal strips, blocked and each strip was incubated with anti-pY466 cortactin, anti-pY416 SFK or anti-actin. (C) pSFK (Y416) and (D) pCort (Y466), cortactin phosphorylated by Src at the Y 466 residue. Actin was used for protein loading normalization. Actin labeling is duplicated in (C,D) . pSFK/Act = densitometric rate of pSFK over actin. pCort/Act = densitometric rate of pCort over actin. These are representative observations from at least three independent experiments.

Article Snippet: Rabbit anti-pT202/Y204 ERK1/2 (#9101S) and rabbit anti-pY416 SFK (#2101S) were obtained from Cell Signaling, Beverly, MA, USA.

Techniques: Activation Assay, Phospho-proteomics, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Membrane, Stripping Membranes, Residue, Labeling

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Inhibition of cell-matrix adhesions prevents cartilage chondrocyte death following impact injury.

doi: 10.1002/jor.22523

Figure Lengend Snippet: Figure 3. Confocal microscopy and cell viability immediately after impact injury. (A) Confocal micrographs show live (green) and dead (red) chondrocytes in an impact site in an un-treated control explant, and in explants treated with 10 mM SFKi and either 10 or 100 mM FAKi. Compared to control, fewer dead chondrocytes were observed in SFKs or FAKi treated groups. (B) Statistical analysis revealed that chondrocyte viability was significantly higher in SFKs or FAKi treated explants compared to control. Between two tested concentrations, 100 mM FAKi was more effective than 10 mM. Asterisk represents statistically significant (p < 0.05, p < 0.01). Bars ¼ 500 mm.

Article Snippet: Published by Wiley Periodicals, Inc. 448 JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2014 After 2 days, the explants were randomly distributed and were treated with fresh culture medium containing 10 or 100mM focal adhesion kinase inhibitor (FAKi) (Santa Cruz Biotechnology, Dallas, TX) to block phosphorylation of FAK at the kinase domain (Try 397) or were treated with fresh culture medium containing 10mM Src family kinase inhibitor (SFKi) (Selleckchem, Houston, TX) to block phosphorylation of SFKs at kinase domain (Tyr 416) for 2h.

Techniques: Confocal Microscopy, Control

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Inhibition of cell-matrix adhesions prevents cartilage chondrocyte death following impact injury.

doi: 10.1002/jor.22523

Figure Lengend Snippet: Figure 5. Kinetics of FAK and SFKi by western blot analysis. (A) Immunoblot analysis showed that treatment with 10 ng/ml IL-1b and 100 ng/ml TNF-a for 30 min significantly increased SFKs phosphorylation at Tyr 416. 0.1, 1, and 10 mM SFKi diminished this response dose dependently. (B) Analysis of the integrated densities of the bands with the phosphor- to total SFKs ratio. C represents untreated control and Cþ represents 10 ng/ml IL-1b and 100 ng/ml TNF-a treated only. (C) Immunoblots show that treatment with 100 nM fMLF for 30 min did not enhance FAK phosphorylation at Tyr 397; however 100 mM FAKi significantly reduced FAK phosphorylation among tested concentrations of 1, 10, and 100 mM. (D) Analysis of the integrated densities of the bands with the phosphor- to total FAK ratio. C represents untreated control and Cþ represents 100 nM fMLF treated only.

Article Snippet: Published by Wiley Periodicals, Inc. 448 JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2014 After 2 days, the explants were randomly distributed and were treated with fresh culture medium containing 10 or 100mM focal adhesion kinase inhibitor (FAKi) (Santa Cruz Biotechnology, Dallas, TX) to block phosphorylation of FAK at the kinase domain (Try 397) or were treated with fresh culture medium containing 10mM Src family kinase inhibitor (SFKi) (Selleckchem, Houston, TX) to block phosphorylation of SFKs at kinase domain (Tyr 416) for 2h.

Techniques: Western Blot, Phospho-proteomics, Control

Journal: Cell Adhesion & Migration

Article Title: Fibronectin induces macrophage migration through a SFK-FAK/CSF-1R pathway

doi: 10.1080/19336918.2016.1221566

Figure Lengend Snippet: Mechanism of FN-induced FAK activation in BAC1.2F5 cells. Actin polymerization is necessary for FN-induced FAK phosphorylation. (A) Cells were cultured in the absence of CSF-1 for 18 hours and then kept in suspension in the presence or not of cytochalasin D (CD, 10 μM) before being plated on FN- or PL-coated (10 μg/ml) dishes for the indicated times. Protein lysates were subjected to immunoprecipitation (IP) and immunoblotting with the indicated antibodies. Representative images of WB from one out of 3 independent experiments are shown. Molecular weight markers are reported on the left of gels. SFK mediate FN-induced FAK phosphorylation. (B) Cells were cultured in the absence of CSF-1 for 18 hours and then kept in suspension for 45 minutes in the presence or the absence of the SFK inhibitor PP2 (10 μM) before being plated on FN- or PL-coated (10 μg/ml) dishes for the indicated times. Protein lysates were subjected to immunoprecipitation (IP) and/or immunoblotting with the indicated antibodies. Representative images of WB from one out of 3 independent experiments are shown. Molecular weight markers are reported on the left of gels. SFK mediate FN-induced macrophage migration. (C) Cells were cultured in the absence of CSF-1 for 18 hours and then treated or not with the SFK inhibitors PP2 or SU6656 (10 μM) for 30 minutes before being subjected to migration assay toward DMEM containing or not FN (10 μg/ml). Migrated cells were counted. Histograms represent means ± SEM of data from 2 independent experiments performed in triplicate. Student's t test: *, p < 0.05; ns, not significant.

Article Snippet: The inhibitors used (substrate; time of pretreatment; manufacturer) were: PP2 and SU6656 42,43 (SFK; 30 minutes; Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), GW2580 44 (CSF-1R; 30 minutes; BioVision, Milpitas, CA, USA), PF-573228 45 (FAK, 30 minutes; MedChem Express).

Techniques: Activation Assay, Phospho-proteomics, Cell Culture, Suspension, Immunoprecipitation, Western Blot, Molecular Weight, Migration

Journal: Cancer Medicine

Article Title: Src controls castration recurrence of CWR22 prostate cancer xenografts

doi: 10.1002/cam4.144

Figure Lengend Snippet: Src inhibitors decrease the frequency and rate of CWR22 recurrence. (A) Schematic view of the treatment protocol in regard to CWR22 androgen-dependent (AD) and castration-recurrent (CR) growth. CWR22 xenograft cells are implanted in castrated, T-pelleted male nude mice, and after the tumor reaches a volume of roughly 250 mm 3 , the pellet is removed (“Castration”). Most tumors either regress somewhat or stop growing over the next month, a period in which the mice were either treated with dasatinib, KXO1 or vehicle. Non-recurrence was defined as no net tumor growth in a 7-month period, based on the regressed tumor size following castration. Recurrence was defined as progressive tumor growth within 7 months of castration to sizes equal to or greater than those at precastration levels. With no treatment or vehicle, 40–50% of the tumors started to recur between 3–5 postcastration. (B) Frequency and rate of CWR22 recurrence postcastration (T-pellet removal from castrated mice) recurrence in vehicle, dasatinib or KXO1-treated groups ( n = 20).

Article Snippet: Additionally, KXO1 (also called KX2-391) targets several SFK members as well as Abl, and newer data indicate that it also targets tubulin polymerization (D. Hangauer, Kinex Pharmaceuticals LLC, pers. comm.).

Techniques:

Journal: Cancer Medicine

Article Title: Src controls castration recurrence of CWR22 prostate cancer xenografts

doi: 10.1002/cam4.144

Figure Lengend Snippet: Effect of KXO1 1 and dasatinib 2 on tumor occurrence.

Article Snippet: Additionally, KXO1 (also called KX2-391) targets several SFK members as well as Abl, and newer data indicate that it also targets tubulin polymerization (D. Hangauer, Kinex Pharmaceuticals LLC, pers. comm.).

Techniques:

Journal: Cancer Medicine

Article Title: Src controls castration recurrence of CWR22 prostate cancer xenografts

doi: 10.1002/cam4.144

Figure Lengend Snippet: Effect of dasatinib or KXO1 on Src activation levels in vitro and in vivo. (A) Lysates of CWR22Rv1 or CWR22PC cells grown in media with 10% fetal calf serum (fCS) and treated for 24 h with vehicle (“0”), dasatinib or KXO1 (0.5 or 1 μmol/L) were immunoblotted for total Src, Src poY416 , Paxillin, Paxillin poY188 or GAPDH (as a protein loading control). NS, nonspecific band. (B) Cells grown for 72 h in 5% charcoal-stripped serum (CSS) were treated with 10 nmol/L dihydrotestosterone (DHT) (24 h) or 10% fCS (1 h), and lysates were then immunoblotted as in panel (A). (C) T-pelleted nude male mice carrying CWR22 tumors or castrated nude mice carrying CWR22Rv1 tumors were treated for 14 days with vehicle (V), dasatinib (D) or KXO1 (K), and then tumors were excised 3 h after the last treatment. Tumor lysates were analyzed for total- or poY416-Src as in panel (A). (D) Castration-recurrent prostate cancer (CR-CaP) tumors arising from mice treated for 28 days right after castration with dasatinib (tumors 11–15), KXO1 (tumors 16–19) or vehicle (tumors 1–10), as described in Table , were analyzed for Src and Src poY416 by immunoblot as above.

Article Snippet: Additionally, KXO1 (also called KX2-391) targets several SFK members as well as Abl, and newer data indicate that it also targets tubulin polymerization (D. Hangauer, Kinex Pharmaceuticals LLC, pers. comm.).

Techniques: Activation Assay, In Vitro, In Vivo, Control, Western Blot

Journal: Cancer Medicine

Article Title: Src controls castration recurrence of CWR22 prostate cancer xenografts

doi: 10.1002/cam4.144

Figure Lengend Snippet: Growth inhibitory concentrations (GI 50 1 ) of KXO1 and dasatinib.

Article Snippet: Additionally, KXO1 (also called KX2-391) targets several SFK members as well as Abl, and newer data indicate that it also targets tubulin polymerization (D. Hangauer, Kinex Pharmaceuticals LLC, pers. comm.).

Techniques: