PMC

An overview scheme of COG112 inhibition of SET functions. (A) SET functions to inhibit both PP2A and nm23-H1. SET is also required for Rac1-mediated cell migration. (B) In the presence of COG112, SET is unable to form protein complexes with PP2A-c, nm23-H1 and Rac1. We conclude that COG112 inhibits multiple oncoprotein functions of SET in human cancer cell lines.

(A) Co-imunoprecipitation experiments show that COG112 pretreatment inhibits SET/Rac1 association in EGF-stimulated MDA-MB-231 cells. (B) Active Rac1-GTPase was pulled-down using GST-PAK1-p21 protein binding domain fusion protein with glutathione-conjugated beads from COG112 pre-treated MDA-MB-231 cells. COG112 caused a concentration-dependent decrease in activated Rac1-GTPase in response to EGF. MDA-MB-231 cells were incubated with COG112 and allowed to (C) migrate or (D) invade using 5% FBS as a chemotractant for 18 hours. Migrating/invading cells were fixed, stained and counted. Data represent mean values (± SD) of cell counts from three independent experiments.

Serum-starved MDA-MB-231 cells were exposed to COG112 for 2 h and stimulated with EGF. (A) Western blot analysis of Akt activation in response to COG112 and EGF in the presence or absence of OA. (B) COG112 inhibits Akt activation in a concentration- and OA- dependent manner. Densitometry analysis of Akt (ser-473) phosphorylation versus COG112 is shown ± OA. Data represent mean ± SEM and significance was determined by paired t test. (C) Representative western blot and (D) densitometry analysis of EGFR and PDK1 activation in response to COG112 and EGF. Data represent mean values (± SEM) of the relative densitometry of phospho-protein:total protein from three independent experiments. Significance is relative to EGF controls.

(A) COG112 interacts with SET. MDA-MB-231 whole cell lysate was incubated with biotin or biotin-COG112. Proteins pulled-down with streptavidin-agarose beads were immunobloted with anti-SET/I₂PP2A. (B) COG112 inhibits SET:PP2A-c association. MDA-MB-231 cells were incubated with COG112 for 2 hr prior to EGF. SET and PP2A-c protein complexes were immunoprecipitated and analyzed for SET and PP2A-c co-precipitation. COG112 inhibited SET:PP2A-c complex formation in a concentration-dependent manner. (Inset: SET:PP2A-c association in serum starved cells treated with or without EGF) (C) COG112 increases PP2A activity. PP2A-c was immunoprecipitated from COG112 treated U87 cells and phosphatase activity measured using a synthetic phospho-peptide. Data represent mean values (± SEM) of released phosphate from three independent experiments. Significance is relative to EGF controls.

Treating MDA-MB-231 cells with COG112 for 3 hr caused SET to dissociate from nm23-H1. (A) Representative co-immunoprecipitation experiment showing reduced SET bound to nm23-H1 in COG112 treated cells. (B) Densitometry analysis of the SET:nm23-H1 complex in response to COG112. Data represent mean values (± SD) of relative SET:nm23-H1 densitometry from three independent experiments. Significance is relative to untreated controls. (B) Western blot of nuclear extract showing that COG112 caused the nuclear accumulation of nm23-H1. (C) Fractionated MDA-MB-231 cells were incubated with COG112 for 3 hr. COG112 resulted in the nuclear accumulation of nm23-H1. (D) MDA-MB-231 cells were treated with COG112 (1 µM) for indicated times and nm23-H1 levels were determined from fractionated cells. (E) COG112 increased the nuclear exonuclease activity of MDA-MB-231 cells. SHS oligonucleotides were incubated with nuclear protein from treated cells; DNA cleavage products are shown from a representative experiment.

Serum-starved U87 cells were exposed to COG112 for 2 h and stimulated with EGF. Representative western blots of Akt substrates (A) mTOR (phosphoserine 2448), (B) GSK-3b (phosphoserine 9) show that COG112 inhibits Akt signaling but not in the presence of OA. (C) c-Myc protein levels are similarly down-regulated by COG112 in an OA-sensitive manner. (D) Nuclear protein from COG112 treated U87 cells were assayed for c-Myc binding to its consensus sequence DNA using an ELISA based kit. Data represent mean values (± SD) of relative c-Myc-DNA levels from three independent experiments. Significance is relative to untreated controls. (E) The effect of COG112 on Detroit 551 fibroblasts, MDA-MB-231 and U87 cell proliferation in serum-containing media. Data represent mean values (± SEM) and significance is relative to FBS positive control.