Serine phosphorylation of the RhoGEF Trio stabilizes endothelial cell-cell junctions.

The RhoGEF Trio is a large multi-domain protein and an activator of the small GTPases Rac1, RhoG, and RhoA. Although Trio has been implicated in many cellular mechanisms like leukocyte transendothelial migration, cell-cell junction stability, lamellipodia formation, axon outgrowth, and muscle fusion, it remains unclear how Trio is activated. Using stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry analysis of endothelial cells, we identified two serine residues (S1785/S1786) located in between the two exchange domains of Trio that were highly phosphorylated upon short thrombin treatment. Using phosphomimetic Trio S1785D/S1786D double mutants, we did not find an increase in Rac1/RhoG activity, indicating that the phosphorylation events do not increase Trio exchange activity. However, we found that the Trio mutants localized more strongly at cell-cell junctions and prevented junction destabilization upon thrombin treatment, judged by junction linearity. Our data suggest that serine phosphorylation of Trio potentiates the localization of Trio to junctional regions, resulting in locally promoting the exchange for Rac1 at junction regions and increasing endothelial cell-cell junction stability upon permeability-inducing reagents such as thrombin.

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/ß as downstream targets of IGF1R signaling, which are independent from PI3K/ATK and MAPK/ERK pathways. PAK2 and PIXα/ß modulate IGF1R signaling-driven cell scattering. Targeting PIXα/ß 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.

Epidermal growth factor receptor signalling in human breast cancer cells operates parallel to estrogen receptor α signalling and results in tamoxifen insensitive proliferation.

BACKGROUND: Tamoxifen resistance is a major problem in the treatment of estrogen receptor (ER) α -positive breast cancer patients. Although the mechanisms behind tamoxifen resistance are still not completely understood, clinical data suggests that increased expression of receptor tyrosine kinases is involved. Here, we studied the estrogen and anti-estrogen sensitivity of human breast cancer MCF7 cells that have a moderate, retroviral-mediated, ectopic expression of epidermal growth factor receptor (MCF7-EGFR). METHODS: Proliferation of MCF7-EGFR and parental cells was induced by 17ß-estradiol (E2), epidermal growth factor (EGF) or a combination of these. Inhibition of proliferation under these conditions was investigated with 4-hydroxy-tamoxifen (TAM) or fulvestrant at 10(-12) to 10(-6) M. Cells were lysed at different time points to determine the phosphorylation status of EGFR, MAPK1/3, AKT and the expression of ERα. Knockdown of target genes was established using smartpool siRNAs. Transcriptomics analysis was done 6 hr after stimulation with growth factors using Affymetrix HG-U133 PM array plates. RESULTS: While proliferation of parental MCF7 cells could only be induced by E2, proliferation of MCF7-EGFR cells could be induced by either E2 or EGF. Treatment with TAM or fulvestrant did significantly inhibit proliferation of MCF7-EGFR cells stimulated with E2 alone. EGF treatment of E2/TAM treated cells led to a marked cell proliferation thereby overruling the anti-estrogen-mediated inhibition of cell proliferation. Under these conditions, TAM however did still inhibit ERα- mediated transcription. While siRNA-mediated knock-down of EGFR inhibited the EGF- driven proliferation under TAM/E2/EGF condition, knock down of ERα did not. The TAM resistant cell proliferation mediated by the conditional EGFR-signaling may be dependent on the PI3K/Akt pathway but not the MEK/MAPK pathway, since a MEK inhibitor (U0126), did not block the proliferation. Transcriptomic analysis under the various E2/TAM/EGF conditions revealed that E2 and EGF dependent transcription have little overlap and rather operate in a parallel fashion. CONCLUSIONS: Our data indicate that enhanced EGFR-driven signalling is sufficient to overrule the TAM- mediated inhibition of E2-driven cell proliferation. This may have profound implications for the anti-estrogen treatment of ER-positive breast cancers that have increased levels of EGFR.

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.