Simultaneous inhibition of the HGF/MET and Erk1/2 pathways affect uveal melanoma cell growth and migration

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1. MET mRNA expression levels in GNAQ mutant and wild-type uveal melanoma cells.

(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 GNAQ mutant and wild-type uveal melanoma cells. MET protein expression (top panel) and actin protein expression (lower panel) as determined by western-blot.

Figure 2. Effect of METi (MK-8033) treatment on MET phosphorylation in GNAQ mutant and wild-type uveal melanoma cells.

(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 GNAQ 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 GNAQ 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 GNAQ mutant uveal melanoma cells (MEL202).

Figure 3. Effect of METi treatment on Erk1/2 and AKT phosphorylation in GNAQ mutant and wild-type uveal melanoma cells following stimulation with HGF (100/ng/ml).

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.

Figure 4. Effect of MEK and/or MET inhibition on the growth of GNAQ mutant versus wild-type uveal melanoma cells.

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

Figure 5. Effect of MEKi and/or METi treatment on PARP cleavage in uveal melanoma cells.

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

Figure 6. Effect of METi treatment on GNAQ mutant or wild-type uveal melanoma cell migration.

(A) Representative images of distinct GNAQ 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 GNAQ wild-type or mutant uveal melanoma cells that migrated following 0, 1 and 2.5 µM METi treatment.