Twist1 Promotes Gastric Cancer Cell Proliferation through Up-Regulation of FoxM1

<div><p>Twist-related protein 1 (Twist1), also known as class A basic helix-loop-helix protein 38 (bHLHa38), has been implicated in cell lineage determination and differentiation. Previous studies demonstrate that Twist1 expression is up-regulated in gastric cancer with poor clinical outcomes. Besides, Twist1 is suggested to be involved in progression of human gastric cancer. However, its biological functions remain largely unexplored. In the present study, we show that Twist 1 overexpression leads to a significant up-regulation of FoxM1, which plays a key role in cell cycle progression in gastric cancer cells. In contrast, knockdown of Twist 1 reduces FoxM1 expression, suggesting that FoxM1 might be a direct transcriptional target of Twist 1. At the molecular level, we further reveal that Twist 1 could bind to the promoter region of FoxM1, and subsequently recruit p300 to induce FoxM1 mRNA transcription. Therefore, our results uncover a previous unknown Twist 1/FoxM1 regulatory pathway, which may help to understand the mechanisms of gastric cancer proliferation.</p> </div

FoxM1 expression in gastric cancer cells depletion of Twist 1.

<div><p>(A-B) Quantitative real-time PCR (A) and western blot (B) analysis of FoxM1 expression in NCI-N87 cells transfected with siRNA oligos targeting Twist 1 or negative control siRNA (Ctrl).</p> <p>(C-D) Quantitative real-time PCR (C) and western blot (D) analysis of FoxM1 expression in NCI-N87 cells transfected with siRNA oligos targeting Twist 1 or negative control siRNA (Ctrl).</p></div

Twist 1 depletion inhibits gastric cell proliferation.

<div><p>(A-D) Quantitative real-time PCR and western blot analysis of Twist 1 expression in NCI-N87 (A-B) or AGS (C-D) cells transfected with siRNA oligos targeting Twist 1 or negative control siRNA (Ctrl).</p> <p>(E-F) The growth curve of NCI-N87 (E) or AGS (F) cells cells transfected with siRNA oligos targeting Twist 1 or negative control siRNA (Ctrl).</p> <p>(G-H) The cell proliferative potential (BrdU) was determined in NCI-N87 (G) or AGS (H) cells transfected with siRNA oligos targeting Twist 1 or negative control siRNA (Ctrl).</p> <p>(I-J) The cell cycle phase of NCI-N87 (I) and AGS (J) cells transfected with siRNA oligos targeting Twist 1 or scramble siRNA (Ctrl) were analyzed by flow cytometry. </p></div

Twist 1 overexpression promotes gastric cell proliferation.

<div><p>(A-B) Representative western blot analysis of Twist 1 expression in NCI-N87 (A) or AGS (B) cells transfected with adenoviruses expressing empty vector (EV) or Twist 1.</p> <p>(C-D) The growth curve of NCI-N87 (C) or AGS (D) cells cells transfected with empty vector (EV) or Twist 1.</p> <p>(E-F) The cell proliferative potential (BrdU) was determined in NCI-N87 (E) or AGS (F) cells transfected with empty vector (EV) or Twist 1.</p> <p>(G-H) The cell cycle phase of NCI-N87 (G) and AGS (H) cells transfected with empty vector (EV) or Twist 1 was analyzed by flow cytometry. Cells were labeled for 15 min with PI and immediately analyzed by flow cytometry. Histograms represent the percentage of cells in each phase of the cell cycle (G0/G1, S and G2/M).</p></div

FoxM1 expression in gastric cancer cells overexpressing Twist 1.

<div><p>(A-B) Quantitative real-time PCR (A) and western blot (B) analysis of FoxM1, FoxO1, Stat3, p53 and E2F1 expression in NCI-N87 cells transfected with adenoviruses expressing empty vector (EV) or Twist 1.</p> <p>(C-D) Quantitative real-time PCR (C) and western blot (D) analysis of Twist 1 expression in NCI-N87 cells transfected with adenoviruses expressing empty vector (EV) or Twist 1.</p></div

Twist 1 silencing on the regulation of cell-cycle regulators.

<p>(A-D) Quantitative real-time PCR and western blot analysis of p21, p27, Cyclin B1, Cyclin D1, Cyclin D2 and Cyclin E in NCI-N87 (A-B) or AGS (C-D) cells transfected with siRNA oligos targeting Twist 1 or negative control siRNA (Ctrl).</p

Twist 1 overexpression on the regulation of cell-cycle regulators.

<p>(A-D) Quantitative real-time PCR and western blot analysis of p21, p27, Cyclin B1, Cyclin D1, Cyclin D2 and Cyclin E in NCI-N87 (A-B) or AGS (C-D) cells transfected with adenoviruses expressing empty vector (EV) or Twist 1.</p

Twist 1 promotes FoxM1 promoter activity through recruiting p300.

<div><p>(A) Diagram of the Twist 1-binding site in the human FoxM1 promoter (1kb) and two FoxM1 luciferase reporters. WT-Luc: wild-type luciferase reporter; Mut-Luc: luciferase reporter carrying point mutations of Twist 1 binding site. Mutations were underlined.</p> <p>(B) Activation of FoxM1 luciferase reporters by Twist 1 in NCI-N87 cells.</p> <p>(C) The binding of Twist 1 on the promoter regions of human FoxM1 gene were analyzed by ChIP assays and quantified by real-time PCR. The region containing -2000 to -1800 bp were used as a negative control. </p> <p>(D) Co-immunoprecipitation of p300 and Twist 1 in NCI-N87 cells.</p> <p>(E) Activation of FoxM1 luciferase reporters by Twist 1 and p300 in NCI-N87 cells.</p> <p>(F) Representative western blot analysis of p300 expression in NCI-N87 cells transfected with siRNA targeting p300 or negative control (Ctrl).</p> <p>(G-H) Quantitative real-time PCR (G) and western blot (H) analysis of FoxM1 expression in NCI-N87 cells transfected with adenoviruses expressing empty vector (EV) or Twist 1. Cells were pre-transfected with siRNA oligos targeting p300 or negative control siRNA (Ctrl) for 24 hours.</p></div

RhlA Exhibits Dual Thioesterase and Acyltransferase Activities during Rhamnolipid Biosynthesis

Rhamnolipids (RLs) are a desirable class of biosurfactants originating from Pseudomonas aeruginosa. Rhamnosyltransferase 1 chain A (RhlA) catalyzes the formation of β-3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) to constitute the RL lipid moiety, and the molecular structure of this moiety exerts major impacts on the physiochemical and biological properties of corresponding RLs. However, the catalytic mechanism and sequence-structure–function relationship of RhlA remain elusive. Here, we report the X-ray crystal structure of P. aeruginosa RhlA with an α/β-hydrolase fold and a canonical nucleophile/histidine/acidic catalytic triad. Unexpectedly, free 3-hydroxy fatty acids within a secondary ligand-binding pocket were observed in the crystal of RhlA, which is traditionally considered an acyltransferase that acts only on acyl carrier protein (ACP)-bound substrates. In vitro isotopic labeling, enzyme kinetics experiments, and QM/MM simulations confirmed that free β-hydroxy fatty acids are a reaction intermediate during HAA synthesis. Moreover, first-shell residue mutations that targeted different ligand-binding pockets resulted in distinct modulation patterns for the two acyl chain lengths of HAAs. In conclusion, the revealed biosynthetic mechanism may guide future engineering for the biosynthesis of designer RLs