IGF1R signaling drives antiestrogen resistance through PAK2/PIX activation in luminal breast cancer

Antiestrogen resistance in estrogen receptor positive (ER+) breast cancer is associated with increased expression and activity of insulin-like growth factor 1 receptor (IGF1R). Here, a kinome siRNA screen has identified 10 regulators of IGF1R-mediated antiestrogen with clinical significance. These include the tamoxifen resistance suppressors BMPR1B, CDK10, CDK5, EIF2AK1, and MAP2K5, and the tamoxifen resistance inducers CHEK1, PAK2, RPS6KC1, TTK, and TXK. The p21-activated kinase 2, PAK2, is the strongest resistance inducer. Silencing of the tamoxifen resistance inducing genes, particularly PAK2, attenuates IGF1R-mediated resistance to tamoxifen and fulvestrant. High expression of PAK2 in ER+ metastatic breast cancer patients is correlated with unfavorable outcome after first-line tamoxifen monotherapy. Phospho-proteomics has defined PAK2 and the PAK-interacting exchange factors PIXα/β as downstream targets of IGF1R signaling, which are independent from PI3K/ATK and MAPK/ERK pathways. PAK2 and PIXα/β modulate IGF1R signaling-driven cell scattering. Targeting PIXα/β entirely mimics the effect of PAK2 silencing on antiestrogen re-sensitization. These data indicate PAK2/PIX as an effector pathway in IGF1R-mediated antiestrogen resistance

The N-Terminal DH-PH Domain of Trio Induces Cell Spreading and Migration by Regulating Lamellipodia Dynamics in a Rac1-Dependent Fashion

The guanine-nucleotide exchange factor Trio encodes two DH-PH domains that catalyze nucleotide exchange on Rac1, RhoG and RhoA. The N-terminal DH-PH domain is known to activate Rac1 and RhoG, whereas the C-terminal DH-PH domain can activate RhoA. The current study shows that the N-terminal DH-PH domain, upon expression in HeLa cells, activates Rac1 and RhoG independently from each other. In addition, we show that the flanking SH3 domain binds to the proline-rich region of the C-terminus of Rac1, but not of RhoG. However, this SH3 domain is not required for Rac1 or RhoG GDP-GTP exchange. Rescue experiments in Trio-shRNA-expressing cells showed that the N-terminal DH-PH domain of Trio, but not the C-terminal DH-PH domain, restored fibronectin-mediated cell spreading and migration defects that are observed in Trio-silenced cells. Kymograph analysis revealed that the N-terminal DH-PH domain, independent of its SH3 domain, controls the dynamics of lamellipodia. Using siRNA against Rac1 or RhoG, we found that Trio-D1-induced lamellipodia formation required Rac1 but not RhoG expression. Together, we conclude that the GEF Trio is responsible for lamellipodia formation through its N-terminal DH-PH domain in a Rac1-dependent manner during fibronectin-mediated spreading and migration

Search for Supersymmetry with Gauge-Mediated Breaking in Diphoton Events with Missing Transverse Energy at CDF II

accepted to Phys. Rev. LettWe present the results of a search for supersymmetry with gauge-mediated breaking and \NONE\to\gamma\Gravitino in the $\gamma\gamma$+missing transverse energy final state. In 2.6$\pm$0.2 \invfb of $p{\bar p}$ collisions at $\sqrt{s}$$=$1.96 TeV recorded by the CDF II detector we observe no candidate events, consistent with a standard model background expectation of 1.4$\pm$0.4 events. We set limits on the cross section at the 95% C.L. and place the world's best limit of 149\gevc on the \none mass at $\tau_{\tilde{\chi}_1^0}$$We present the results of a search for supersymmetry with gauge-mediated breaking and χ˜10→γG˜ in the γγ+missing transverse energy final state. In 2.6±0.2  fb-1 of pp̅ collisions at √s=1.96  TeV recorded by the CDF II detector we observe no candidate events, consistent with a standard model background expectation of 1.4±0.4 events. We set limits on the cross section at the 95% C.L. and place the world’s best limit of 149  GeV/c2 on the χ˜10 mass at τχ˜10≪1  ns. We also exclude regions in the χ˜10 mass-lifetime plane for τχ˜10≲2  ns.Peer reviewe

Measurements of branching fraction ratios and CP asymmetries in B+/- ->D_CP K+/- decays in hadron collisions

We reconstruct B+/- -> D K+/- decays in a data sample collected by the CDF II detector at the Tevatron collider corresponding to 1 fb-1 of integrated luminosity. We select decay modes where the D meson decays to either K- pi+ (flavor eigenstate) or K- K+, pi- pi+ (CP-even eigenstates), and measure the direct CP asymmetry A_CP+ = 0.39 +/- 0.17(stat) +/- 0.04(syst), and the double ratio of CP-even to flavor eigenstate branching fractions R_CP+ = 1.30 +/- 0.24(stat) +/- 0.12(syst). These measurements will improve the determination of the CKM angle gamma. They are performed here for the first time using data from hadron collisions.We reconstruct B±→DK± decays in a data sample collected by the CDF II detector at the Tevatron collider corresponding to 1  fb-1 of integrated luminosity. We select decay modes where the D meson decays to either K-π+ (flavor eigenstate) or K-K+, π-π+ (CP-even eigenstates), and measure the direct CP asymmetry ACP+=0.39±0.17(stat)±0.04(syst), and the double ratio of CP-even to flavor eigenstate branching fractions RCP+=1.30±0.24(stat)±0.12(syst). These measurements will improve the determination of the Cabibbo-Kobayashi-Maskawa angle γ. They are performed here for the first time using data from hadron collisions.Peer reviewe

Inclusive Search for Standard Model Higgs Boson Production in the WW Decay Channel using the CDF II Detector

We present a search for standard model (SM) Higgs boson production using ppbar collision data at sqrt(s) = 1.96 TeV, collected with the CDF II detector and corresponding to an integrated luminosity of 4.8 fb-1. We search for Higgs bosons produced in all processes with a significant production rate and decaying to two W bosons. We find no evidence for SM Higgs boson production and place upper limits at the 95% confidence level on the SM production cross section (sigma(H)) for values of the Higgs boson mass (m_H) in the range from 110 to 200 GeV. These limits are the most stringent for m_H > 130 GeV and are 1.29 above the predicted value of sigma(H) for mH = 165 GeV.We present a search for standard model (SM) Higgs boson production using pp̅ collision data at √s=1.96  TeV, collected with the CDF II detector and corresponding to an integrated luminosity of 4.8  fb-1. We search for Higgs bosons produced in all processes with a significant production rate and decaying to two W bosons. We find no evidence for SM Higgs boson production and place upper limits at the 95% confidence level on the SM production cross section (σH) for values of the Higgs boson mass (mH) in the range from 110 to 200 GeV. These limits are the most stringent for mH>130  GeV and are 1.29 above the predicted value of σH for mH=165  GeV.Peer reviewe

Measurement of the Lambda_b Lifetime in Lambda_b -> Lambda_c+ pi- Decays in p-pbar Collisions at sqrt(s) = 1.96 TeV

Submitted to Phys. Rev. LettWe report a measurement of the lifetime of the Lambda_b baryon in decays to the Lambda_C+ pi- final state in a sample corresponding to 1.1 fb^-1 collected in p-pbar collisions at sqrt(s) = 1.96 TeV by the CDF II detector at the Tevatron collider. Using a sample of about 3000 fully reconstructed Lambda_b events we measure tau(Lambda_b) = 1.401 +- 0.046 (stat) +- 0.035 (syst) ps (corresponding to c.tau(Lambda_b) = 420.1 +- 13.7 (stat) +- 10.6 (syst) um, where c is the speed of light). The ratio of this result and the world average B^0 lifetime yields tau(Lambda_b)/tau(B^0) = 0.918 +- 0.038 (stat and syst), in good agreement with recent theoretical predictions.We report a measurement of the lifetime of the Λb0 baryon in decays to the Λc+π- final state in a sample corresponding to 1.1  fb-1 collected in pp̅ collisions at √s=1.96  TeV by the CDF II detector at the Tevatron collider. Using a sample of about 3000 fully reconstructed Λb0 events we measure τ(Λb0)=1.401±0.046(stat)±0.035(syst)  ps (corresponding to cτ(Λb0)=420.1±13.7(stat)±10.6(syst)  μm, where c is the speed of light). The ratio of this result and the world average B0 lifetime yields τ(Λb0)/τ(B0)=0.918±0.038 (stat) and (syst), in good agreement with recent theoretical predictions.Peer reviewe

Measurement of the Top Quark Mass and ppbar -> ttbar Cross Section in the All-Hadronic Mode with the CDFII Detector

Submitted to Phys. Rev. DWe present a measurement of the top quark mass and of the top-antitop pair production cross section using p-pbar data collected with the CDFII detector at the Tevatron Collider at the Fermi National Accelerator Laboratory and corresponding to an integrated luminosity of 2.9 fb-1. We select events with six or more jets satisfying a number of kinematical requirements imposed by means of a neural network algorithm. At least one of these jets must originate from a b quark, as identified by the reconstruction of a secondary vertex inside the jet. The mass measurement is based on a likelihood fit incorporating reconstructed mass distributions representative of signal and background, where the absolute jet energy scale (JES) is measured simultaneously with the top quark mass. The measurement yields a value of 174.8 +- 2.4(stat+JES) ^{+1.2}_{-1.0}(syst) GeV/c^2, where the uncertainty from the absolute jet energy scale is evaluated together with the statistical uncertainty. The procedure measures also the amount of signal from which we derive a cross section, sigma_{ttbar} = 7.2 +- 0.5(stat) +- 1.0 (syst) +- 0.4 (lum) pb, for the measured values of top quark mass and JES.We present a measurement of the top quark mass and of the top-antitop (tt̅ ) pair production cross section using pp̅ data collected with the CDF II detector at the Tevatron Collider at the Fermi National Accelerator Laboratory and corresponding to an integrated luminosity of 2.9  fb-1. We select events with six or more jets satisfying a number of kinematical requirements imposed by means of a neural-network algorithm. At least one of these jets must originate from a b quark, as identified by the reconstruction of a secondary vertex inside the jet. The mass measurement is based on a likelihood fit incorporating reconstructed mass distributions representative of signal and background, where the absolute jet energy scale (JES) is measured simultaneously with the top quark mass. The measurement yields a value of 174.8±2.4(stat+JES)-1.0+1.2(syst)  GeV/c2, where the uncertainty from the absolute jet energy scale is evaluated together with the statistical uncertainty. The procedure also measures the amount of signal from which we derive a cross section, σtt̅ =7.2±0.5(stat)±1.0(syst)±0.4(lum)  pb, for the measured values of top quark mass and JES.Peer reviewe

Trio regulates Rac1 activity upon FN-mediated cell spreading.

<p>(<b>A</b>) Rac1 activity was measured with biotin-CRIB peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Rac1 activity was increased after 3 h of spreading in shCTRL cells, whereas changes in Rac1 activation were absent in Trio-deficient cells (shTrio). (<b>B</b>) Trio RhoG activity was measured with GST-ELMO as bait (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>). RhoG activity was unaltered in shCTRL and shTrio cells upon cell spreading on FN. (<b>C</b>) Early Rac1 activation upon spreading was affected in Trio-deficient cells as well. Rac1-GTP levels were measured upon 10 or 20 minutes spreading on FN or in suspension as described under A. (<b>D</b>) RhoA activity was measured in the GST-Rhotekin pull-down assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. RhoA activity was high in cells that were in suspension (0 minutes) and decreased upon spreading on FN (180 minutes). No difference between shCTRL and shTrio cells was measured. All experiments described above were carried out at least three times.</p

Trio-D1 binds to the C-terminus of Rac1 but not of RhoG and activates Rac1 independent of its SH3 domain.

<p>(<b>A</b>) HeLa cells were transfected with Myc-Trio-Full-length (FL) and a pull-down experiment with biotin-tagged peptides that encode for the last 10 amino-acids of the C-terminus of Rac1, RhoG and RhoA was performed, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Western blot analysis showed that Trio-FL binds to the Rac1 C-terminus peptide, but not to RhoA or RhoG C-termini. As a control, β-Pix binding to the C-terminus of Rac1, but not of RhoA or RhoG is shown. (<b>B</b>) Myc-Trio-D1 was transfected into HeLa cells, and a peptide pull down was performed as described under A. Western blot analysis showed that Trio-D1 associates with the C-terminus of Rac1, but not with the CTRL or RhoG peptide. Left lane shows Myc-Trio-D1 input. (<b>C</b>) HeLa cells were transfected with GFP-Trio-D1ΔSH3 or GFP-Trio-D1+SH3 constructs and a Rac1 C-terminal peptide pull down was performed. Western blot analysis showed that the C-terminus of Rac1 required the SH3 domain of Trio-D1 to interact. Blots were incubated with an Ab against GFP to stain for Trio constructs. (<b>D</b>) HeLa cells were transfected with GFP-Trio-D1+SH3 constructs and a peptide pull down was performed with biotinylated peptides encoding control sequence, the Rac1 C-terminal domain or the Rac1 C-terminal domains in which the proline stretch had been mutated to alanines (P/A) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#pone.0029912-Nethe1" target="_blank">[10]</a>. Western blot analysis showed that Trio-D1+SH3 required the proline-rich stretch in the Rac1 C-terminus to bind. Blots were incubated with an Ab against GFP to stain for Trio constructs. (<b>E</b>) HeLa cells were transfected with GFP-CAAX, GFP-Trio-D1ΔSH3 or GFP-Trio-D1+SH3 constructs, and Rac1-GTP activity assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Western blot analysis showed that Rac1 is activated by Trio-D1, independent of the SH3 domain (upper panel). (<b>F</b>) HeLa cells were transfected with GFP-CAAX, GFP-Trio-D1ΔSH3 or GFP-Trio-D1+SH3 constructs and a pull-down assay using glutathione-beads to precipitate GST-Rac1-G15A mutants was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#s4" target="_blank">Materials and Methods</a>. Western blot analysis showed that Rac1 needed the SH3 domain of Trio-D1 to interact (upper panel), because the binding was less efficient when Trio-D1 lacked the SH3 domain. Lower panel shows GST-Rac1-G15A input. Lower unidentified band in upper panel is due to GST isolation and a-specific staining of the antibody. Graph below shows the quantification of the binding of GST-Rac1-G15A to Trio-D1ΔSH3 and Trio-D1+SH3. No significant difference was found for the presence of the SH3 domain in the binding to GST-Rac1-G15A. Experiment was carried out three times, independently from each other. Data are mean ± SEM. ns: not significant.</p

Trio induces membrane ruffles.

<p>(<b>A</b>) Schematic overview of the Trio protein (3097 amino acids, molecular weight approximately 350 kDa), indicating in green the N-terminal DH-PH unit including an SH3 domain and in red the C-terminal DH-PH unit. The third catalytic domain of Trio is a kinase domain (yellow). At the N-terminus, a Sec14 domain and spectrin repeats are present. Below the GFP/Myc-tagged constructs used in this manuscript are depicted: Trio-D1 encodes for the N-terminal DH-PH domain including the flanking SH3 domain, Trio-D1ΔSH3 domain represents the N-terminal DH-PH domain lacking the SH3 domain, and Trio-D2 representing the C-terminal DH-PH domain. (<b>B</b>) HeLa cells were cultured on glass cover slips and transfected as indicated with GFP-tagged constructs. Immunofluorescent imaging showed that GFP did not affect the morphology of the cells. GFP-Trio full length (FL) and GFP-Trio-D1 induced lamellipodia (arrowheads) and co-localized with F-actin (red), as is shown in the merge images. For the Trio-FL, 68%±7 of the transfected cells induced lamellipodia as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029912#pone-0029912-g001" target="_blank">figure 1B</a>. For Trio-D1, 79%±4 of the transfected cells induced lamellipodia. GFP-Trio-D2 in green induced stress fibers (arrowheads), shown by F-actin staining in red. 46%±12 of these transfected cells i9nduced stress fibers, as shown. Data are mean ± SEM. Bar, 20 µm. Images at the right show merged magnification of F-actin in red and GFP-tagged protein in green. Bar, 10 µm. (<b>C</b>) Changes in morphology analyzed by scanning electron microscopy. No change in morphology is observed at the periphery or surface of GFP-expressing HeLa cells (arrowheads), whereas Trio-D1 induced large dorsal and lateral lamellipodia (arrowheads). Bar, 50 µm. Image on the right shows a magnification (Zoom) of Trio-D1-induced dorsal lamellipodia (arrowheads). Bar, 5 µm. Two lower images show lamellipodia (arrowheads), induced by a constitutively active form of RhoG (Q61L) (left image) and Rac1 (Q61L) (right image), both comparable with the lamellipodia induced by Trio-D1. Bar, 10 µm.</p