Targeting SET/I(2)PP2A oncoprotein functions as a multi-pathway strategy for cancer therapy

Abstract

The SET oncoprotein participates in cancer progression by affecting multiple cellular processes, inhibiting the tumor suppressor protein phosphatase 2A (PP2A), and inhibiting the metastasis suppressor nm23-H1. On the basis of these multiple activities, we hypothesized that targeted inhibition of SET would have multiple discrete and measurable effects on cancer cells. Here, the effects of inhibiting SET oncoprotein function on intracellular signaling and proliferation of human cancer cell lines was investigated. We observed the effects of COG112, a novel SET interacting peptide, on PP2A activity, Akt signaling, nm23-H1 activity and cellular migration/invasion in human U87 glioblastoma and MDA-MB-231 breast adenocarcinoma cancer cell lines. We found that COG112 interacted with SET protein and inhibited the association between SET and PP2A catalytic subunit (PP2A-c) and nm23-H1. The interaction between COG112 and SET caused PP2A phosphatase and nm23-H1 exonuclease activities to increase. COG112-mediated increases in PP2A activity resulted in the inhibition of Akt signaling and cellular proliferation. Additionally, COG112 inhibited SET association with Ras-related C(3) botulinum toxin substrate 1 (Rac1), leading to decreased cellular migration and invasion. COG112 treatment releases the SET-mediated inhibition of the tumor suppressor PP2A, as well as the metastasis suppressor nm23-H1. These results establish SET as a novel molecular target and that the inhibition of SET may have beneficial effects in cancer chemotherapy.

Figure 1. COG112 Interacts with SET and Increases PP2A Activity

(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.

Figure 2. COG112 Inhibits Akt Activation via PP2A

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.

Figure 3. PP2A Mediates COG112 Inhibition of Cancer Cell Proliferation

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.

Figure 3. PP2A Mediates COG112 Inhibition of Cancer Cell Proliferation

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.

Figure 4. COG112 Increases nm23-H1 Metastasis Suppressor Activity

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.

Figure 4. COG112 Increases nm23-H1 Metastasis Suppressor Activity

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.

Figure 5. COG112 Inhibits SET/Rac1 Association and Cell Migration

(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.

Figure 6. Targeting SET Oncoprotein Function has Multiple Effects

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.