MET mRNA expression levels in <i>GNAQ</i> mutant and wild-type uveal melanoma cells.

<p>(A) MET mRNA expression (top panel) and actin mRNA expression (lower panel) as determined by RT-PCR. Images are from the same gel at the same exposure. (B) MET protein expression levels in <i>GNAQ</i> mutant and wild-type uveal melanoma cells. MET protein expression (top panel) and actin protein expression (lower panel) as determined by western-blot.</p

Effect of METi (MK-8033) treatment on MET phosphorylation in <i>GNAQ</i> mutant and wild-type uveal melanoma cells.

<p>(A) The upper panel demonstrates the effect of MK-8033 at 0, 1 and 2.5 µM on MET phosphorylation in WT uveal melanoma cells (MEL290), and the lower panel demonstrates the effect of MK-8033 on MET phosphorylation in <i>GNAQ</i> mutant cells (MEL202). Total MET protein expression is shown for each cell line under each condition. (B) Effect of METi (MK-8033) and/or MEKi (AZD6244) treatment on Erk1/2 phosphorylation in <i>GNAQ</i> mutant and wild-type uveal melanoma cells. The upper panel demonstrates the effect of MK-8033 and/or AZD6244 on Erk1/2 phosphorylation in WT uveal melanoma cells (MEL285), and the lower panel demonstrates the effect of MK-8033 and/or AZD6244 on Erk1/2 phosphorylation in <i>GNAQ</i> mutant uveal melanoma cells (MEL202).</p

Effect of METi treatment on Erk1/2 and AKT phosphorylation in <i>GNAQ</i> mutant and wild-type uveal melanoma cells following stimulation with HGF (100/ng/ml).

<p>The effect of HGF on uveal melanoma cell Erk1/2 phosphorylation (upper panel) or AKT phosphorylation (lower panel) with or without METi treatment at 0.25 or 2.5 µM. Images are from the same blot at the same exposure.</p

Effect of METi treatment on <i>GNAQ</i> mutant or wild-type uveal melanoma cell migration.

<p>(A) Representative images of distinct <i>GNAQ</i> wild-type (WT1 =  Mel285, WT2 =  Mel290) or mutant (Mut1 =  Mel270, Mut2 =  Mel 202) uveal melanoma cell line migration, stained following 0, 1 and 2.5 µM METi treatment. (B) Percent of <i>GNAQ</i> wild-type or mutant uveal melanoma cells that migrated following 0, 1 and 2.5 µM METi treatment.</p

Effect of MEK and/or MET inhibition on the growth of <i>GNAQ</i> mutant versus wild-type uveal melanoma cells.

<p>The upper panel shows the effect of 25/or 2.5 µM METi treatment on two distinct <i>GNAQ</i> mutant cells, OMM2.3 (left) and MEL202 (right). The lower panel shows the results of the same treatment conditions in <i>GNAQ</i> wild-type cells, MEL285 (left) and MEL290 (right).</p

Effect of MEKi and/or METi treatment on PARP cleavage in uveal melanoma cells.

<p>The upper panel shows the effect of MEKi at 25/or METi at 2.5 µM on two distinct <i>GNAQ</i> mutant cells, OMM2.5 (left) and MEL202 (right). The lower panel shows the results in <i>GNAQ</i> wild-type cells, MEL285 (left) and MEL290 (right).</p

Simultaneous Inhibition of the HGF/MET and Erk1/2 Pathways Affect Uveal Melanoma Cell Growth and Migration

<div><p>Purpose</p><p>Nearly all primary uveal melanoma (UM) that metastasize involve the liver. Hepatocyte growth factor (HGF) is proposed to be an important microenvironmental element in attracting/supporting UM metastasis through activation of MET. The majority (>85%) of UM express mutations in the G-alpha proteins, that drive the MEK-ERK1/2 pathway. Thus, we proposed that the combination of MET and MEK inhibition would inhibit the growth and migration of G-alpha protein mutant versus non-mutant UM cells.</p><p>Methods</p><p>Western-blots demonstrated the relative protein levels of ERK1/2 and MET in UM cells. Cells were treated with the small molecule inhibitors AZD6244 (MEKi) and/or MK-8033 (METi) and downstream markers evaluated. Further studies determined the effect of combination MEKi and METi treatment on cell growth, apoptosis and migration.</p><p>Results</p><p>All G-alpha protein mutant UM cell lines express MET mRNA and protein. The level of mRNA expression correlates with protein expression. MEKi, but not METi treatment results in markedly reduced ERK1/2 phosphorylation. Either MEKi or METi treatment alone results in reduced cell proliferation, but only modest induction of apoptosis. The combination MEKi+METi results in significant reduction of proliferation in G-alpha protein mutant cells. UM cell migration was blocked by METi, but not MEKi treatment.</p><p>Conclusions</p><p>MET protein expression showed no correlation with G-alpha protein mutation status. Combining MEKi with METi treatment has added benefit to either treatment alone in reducing G-alpha protein mutant UM cell growth. Combining METi with MEKi treatment adds the effect of limiting uveal melanoma cell migration.</p></div

VEGFA expression correlates with drug sensitivity.

<p>Green and red colors represent negative and positive Spearman’s rank correlation coefficients between VEGFA levels and different anticancer drugs’ IC50 in the CCLE (A) and GDSC (B). A positive correlation with IC50 means that the drug is less effective; a negative correlation indicates that it is more effective when VEGFA expression is higher. p < 0.05 correlations are displayed.</p

NQO1 expression correlates with 17-AAG effectiveness in multiple cancer types.

<p>Green color represents negative Spearman’s rank correlation coefficients between NQO1expression and 17-AAG EC50 in the CCLE (A) and IC50 (EC50 was not available) in GDSC (B). These results indicate that 17-AAG works better when NQO1 expression is higher. Only p < 0.05 correlations are shown.</p

Real Time <i>RT-PCR</i> detection of <i>TUSC2</i> gene expression in patients receiving DC-<i>TUSC2</i>.

1<p>Specimens not available.</p