March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
The Protein Kinase C (PKC)/Protein Kinase D (PKD)/Steroid Receptor Coactivator (SRC)-3 pathway is an important therapeutic target in Gα-mutant Uveal Melanomas
Author Affiliations & Notes
  • Vassiliki Poulaki
    Ophthalmology, VA Boston Healthcare System, Boston University, Boston, Massachusetts
  • Sue Anne Chew
    Medicine/Molecular and Cellular Biology,
    Baylor College of Medicine, Houston, Texas
  • Bin He
    Medicine/Molecular and Cellular Biology,
    Baylor College of Medicine, Houston, Texas
  • Vijay Kumar Eedunuri
    Adrienne Helis Malvin Medical Research Foundation, New Orleans, Louisiana
  • Diego Jacinto Bedoya
    Medicine/Molecular and Cellular Biology,
    Baylor College of Medicine, Houston, Texas
  • Martine J. Jager
    Ophthalmology, Leiden University Med Center, Leiden, The Netherlands
  • Bert W. O'Malley
    Molecular and Cellular Biology,
    Baylor College of Medicine, Houston, Texas
  • Nicholas Mitsiades
    Medicine/Molecular and Cellular Biology,
    Baylor College of Medicine, Houston, Texas
  • Footnotes
    Commercial Relationships  Vassiliki Poulaki, None; Sue Anne Chew, None; Bin He, None; Vijay Kumar Eedunuri, None; Diego Jacinto Bedoya, None; Martine J. Jager, None; Bert W. O'Malley, None; Nicholas Mitsiades, None
  • Footnotes
    Support  Adrienne Helis Malvin Medical Research Foundation through its direct engagement in the continuous active conduct of medical research in conjunction with Baylor College of Medicine.
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 6871. doi:
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      Vassiliki Poulaki, Sue Anne Chew, Bin He, Vijay Kumar Eedunuri, Diego Jacinto Bedoya, Martine J. Jager, Bert W. O'Malley, Nicholas Mitsiades; The Protein Kinase C (PKC)/Protein Kinase D (PKD)/Steroid Receptor Coactivator (SRC)-3 pathway is an important therapeutic target in Gα-mutant Uveal Melanomas. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6871.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Activating somatic mutations in the G protein α subunits Gαq and Gα11 (encoded by GNAQ and GNA11, respectively), occur, in a mutually exclusive pattern, in ~80% of uveal melanomas (UMs). As Gα-mutant UMs are resistant to all currently available therapies, including B-Raf inhibitors, novel agents are urgently needed to inhibit Gαq/11 and their downstream signaling pathway in UM. We explored this pathway in UM cell lines in order to identify novel druggable therapeutic targets.

Methods: : Using RNA interference (RNAi), small molecule inhibitors (SMIs) and adenoviral-mediated gene transfer in UM cell lines, we studied the effect of different modulators of the Gαq/11 signaling cascade on cell survival (MTT), oxygen consumption rate (Seahorse XF24 analyzer), mitochondrial membrane potential (JC1), autophagy (detection of LC3BII accumulation by immunoblotting; intracellular localization of LC3B by deconvolution fluorescence microscopy), MEK/ERK signaling and caspase cleavage (immunoblotting).

Results: : GNAQ-mutant (but not BRAF-mutant) UM cell lines were very sensitive to Gαq depletion by RNAi, undergoing early mitochondrial dysfunction and inhibition of MEK/ERK signaling, followed by caspase-independent death exhibiting hallmarks of autophagy (LC3BII accumulation and association with perinuclear autophagosomes). RNAi-based screening for kinase mediators identified PKCα and PKD1 as necessary for MEK/ERK activation and cell viability downstream of Gαq. GNAQ-mutant and GNA11-mutant UM cell lines were also more sensitive than BRAF-mutant cell lines to SMIs of PKC (Bisindolylmaleimide I, Gö6983, rottlerin, midostaurin/PKC412) and PKD (kbNB142-70), again undergoing caspase-independent death (exhibiting hallmarks of autophagy) that was enhanced by concurrent treatment with the MEK inhibitor AZD6244 or the Akt inhibitor MK2206. Depletion of Gαq or PKCα or PKD1 (by RNAi), or SMIs of PKC or PKD, promoted the proteasomal degradation of SRC-3, a potent oncogenic transcription coactivator that is frequently overexpressed in cancer and confers poor prognosis. Depletion of SRC-3 via RNAi induced cell death in Gα-mutant, but not in Gα-wild type UM cell lines. Exogenous overexpression of SRC-3 protected GNAQ-mutant UM cells from cell death induced by GNAQ RNAi or PKC inhibition.

Conclusions: : The PKC/PKD/SRC-3 pathway is a downstream effector of mutant Gα proteins in UM, driving MEK/ERK activation, cell growth and survival. Our results highlight important novel druggable therapeutic targets for UM. In particular, PKC/PKD SMIs and their combinations with MEK and Akt SMIs appear very promising approaches for treatment of Gα-mutant UM.

Keywords: melanoma • apoptosis/cell death • signal transduction 
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