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Destabilisation of dimeric 14-3-3 proteins as a novel approach to anti-cancer therapeutics.

Woodcock JM, Coolen C, Goodwin KL, Baek DJ, Bittman R, Samuel MS, Pitson SM, Lopez AF - Oncotarget (2015)

Bottom Line: Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells.Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice.Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Biology, SA Pathology and University of South Australia, Adelaide, SA, Australia.

ABSTRACT
14-3-3 proteins play a pivotal role in controlling cell proliferation and survival, two commonly dysregulated hallmarks of cancers. 14-3-3 protein expression is enhanced in many human cancers and correlates with more aggressive tumors and poor prognosis, suggesting a role for 14-3-3 proteins in tumorigenesis and/or progression. We showed previously that the dimeric state of 14-3-3 proteins is regulated by the lipid sphingosine, a physiological inducer of apoptosis. As the functions of 14-3-3 proteins are dependent on their dimeric state, this sphingosine-mediated 14-3-3 regulation provides a possible means to target dimeric 14-3-3 for therapeutic effect. However, sphingosine mimics are needed that are not susceptible to sphingolipid metabolism. We show here the identification and optimization of sphingosine mimetics that render dimeric 14-3-3 susceptible to phosphorylation at a site buried in the dimer interface and induce mitochondrial-mediated apoptosis. Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells. Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice. Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery.

No MeSH data available.


Related in: MedlinePlus

A. Structures of the trimethylammonium (TMA) compounds assessed for 14-3-3 modulating activity. B. Phosphorylation of 14-3-3 by PKA in vitro in presence or absence of TMA compounds at the concentrations shown. The upper panel is [32P]-phospho-labeled 14-3-3ζ and the lower panel is Coomassie stained 14-3-3 protein. C. Effect of TMA compounds on Jurkat cell after 20 h treatment at the concentrations shown. Cell viability is shown in open bars and TMRE negative staining cells are shown in black bars. The error bars show the range of duplicate determinations: and the results are representative of multiple experiments.
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Figure 1: A. Structures of the trimethylammonium (TMA) compounds assessed for 14-3-3 modulating activity. B. Phosphorylation of 14-3-3 by PKA in vitro in presence or absence of TMA compounds at the concentrations shown. The upper panel is [32P]-phospho-labeled 14-3-3ζ and the lower panel is Coomassie stained 14-3-3 protein. C. Effect of TMA compounds on Jurkat cell after 20 h treatment at the concentrations shown. Cell viability is shown in open bars and TMRE negative staining cells are shown in black bars. The error bars show the range of duplicate determinations: and the results are representative of multiple experiments.

Mentions: We previously established that non-acylated sphingolipids with a net positive charge are capable of rendering 14-3-3 phosphorylatable [15]. To identify new compounds that are capable of rendering 14-3-3 phosphorylatable but are not susceptible to sphingolipid metabolism, we assessed non-sphingoid cationic lipids such as quaternary ammonium compounds for effects on 14-3-3 phosphorylatability. In our in vitro system using recombinant 14-3-3ζ as substrate and PKA catalytic subunit as the phosphorylating enzyme, we found that trimethylammonium (TMA) molecules with an alkyl chain of 14 carbons or longer rendered 14-3-3 phosphorylatable, whereas molecules with a shorter alkyl chain were ineffective (Figure 1A & 1B).


Destabilisation of dimeric 14-3-3 proteins as a novel approach to anti-cancer therapeutics.

Woodcock JM, Coolen C, Goodwin KL, Baek DJ, Bittman R, Samuel MS, Pitson SM, Lopez AF - Oncotarget (2015)

A. Structures of the trimethylammonium (TMA) compounds assessed for 14-3-3 modulating activity. B. Phosphorylation of 14-3-3 by PKA in vitro in presence or absence of TMA compounds at the concentrations shown. The upper panel is [32P]-phospho-labeled 14-3-3ζ and the lower panel is Coomassie stained 14-3-3 protein. C. Effect of TMA compounds on Jurkat cell after 20 h treatment at the concentrations shown. Cell viability is shown in open bars and TMRE negative staining cells are shown in black bars. The error bars show the range of duplicate determinations: and the results are representative of multiple experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4546484&req=5

Figure 1: A. Structures of the trimethylammonium (TMA) compounds assessed for 14-3-3 modulating activity. B. Phosphorylation of 14-3-3 by PKA in vitro in presence or absence of TMA compounds at the concentrations shown. The upper panel is [32P]-phospho-labeled 14-3-3ζ and the lower panel is Coomassie stained 14-3-3 protein. C. Effect of TMA compounds on Jurkat cell after 20 h treatment at the concentrations shown. Cell viability is shown in open bars and TMRE negative staining cells are shown in black bars. The error bars show the range of duplicate determinations: and the results are representative of multiple experiments.
Mentions: We previously established that non-acylated sphingolipids with a net positive charge are capable of rendering 14-3-3 phosphorylatable [15]. To identify new compounds that are capable of rendering 14-3-3 phosphorylatable but are not susceptible to sphingolipid metabolism, we assessed non-sphingoid cationic lipids such as quaternary ammonium compounds for effects on 14-3-3 phosphorylatability. In our in vitro system using recombinant 14-3-3ζ as substrate and PKA catalytic subunit as the phosphorylating enzyme, we found that trimethylammonium (TMA) molecules with an alkyl chain of 14 carbons or longer rendered 14-3-3 phosphorylatable, whereas molecules with a shorter alkyl chain were ineffective (Figure 1A & 1B).

Bottom Line: Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells.Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice.Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Biology, SA Pathology and University of South Australia, Adelaide, SA, Australia.

ABSTRACT
14-3-3 proteins play a pivotal role in controlling cell proliferation and survival, two commonly dysregulated hallmarks of cancers. 14-3-3 protein expression is enhanced in many human cancers and correlates with more aggressive tumors and poor prognosis, suggesting a role for 14-3-3 proteins in tumorigenesis and/or progression. We showed previously that the dimeric state of 14-3-3 proteins is regulated by the lipid sphingosine, a physiological inducer of apoptosis. As the functions of 14-3-3 proteins are dependent on their dimeric state, this sphingosine-mediated 14-3-3 regulation provides a possible means to target dimeric 14-3-3 for therapeutic effect. However, sphingosine mimics are needed that are not susceptible to sphingolipid metabolism. We show here the identification and optimization of sphingosine mimetics that render dimeric 14-3-3 susceptible to phosphorylation at a site buried in the dimer interface and induce mitochondrial-mediated apoptosis. Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells. Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice. Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery.

No MeSH data available.


Related in: MedlinePlus