Identification of unique MEK-dependent genes in GNAQ mutant uveal melanoma involved in cell growth, tumor cell invasion, and MEK resistance

Abstract

Purpose: Metastatic uveal melanoma represents the most common intraocular malignancy with very poor prognosis and no effective treatments. Oncogenic mutations in the G-protein α-subunit q and 11 have been described in about 85% of uveal melanomas and confer constitutive activation. Multiple signaling pathways are induced as a consequence of GNAQ/11 activation, which include the MEK/ERK kinase cascade. We analyzed the transcriptional profile of cell lines treated with a mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) inhibitor to identify gene targets of activated GNAQ and to evaluate the biologic importance of these genes in uveal melanoma.

Experimental design: We conducted microarray analysis of uveal melanoma cell lines with GNAQ mutations treated with the MEK inhibitor selumetinib. For comparison, we used cells carrying BRAF(V600E) and cells without either mutation. Changes in the expression of selected genes were then confirmed by quantitative real-time PCR and immunoblotting.

Results: We found that GNAQ mutant cells have a MEK-dependent transcriptional output and identified a unique set of genes that are downregulated by MEK inhibition, including the RNA helicase DDX21 and the cyclin-dependent kinase regulator CDK5R1 whereas Jun was induced. We provide evidence that these genes are involved in cell proliferation, tumor cell invasion, and drug resistance, respectively. Furthermore, we show that selumetinib treatment regulates the expression of these genes in tumor tissues of patients with metastatic GNAQ/11 mutant uveal melanoma.

Conclusions: Our findings define a subset of transcriptionally regulated genes by selumetinib in GNAQ mutant cells and provide new insights into understanding the biologic effect of MEK inhibition in this disease.

Figure 1. Selumetinib inhibits cell viability of BRAF^V600E and GNAQ^Q209L/P cell lines

A. Cells with wild-type GNAQ/BRAF (C918, Mel290), mutant GNAQ (92.1, Omm1.3, Mel270, Mel202) and BRAF^V600E (OCM3, OCM1A) were treated with increasing concentrations of selumetinib (0, 25, 50, 100, 250, 500, 1000nM), and analyzed for cell viability on day 5, calculated as percent of untreated controls. Data are representative of three independent experiments. Bars, mean s.d. B. Immunoblots of pERK and pMEK in response to increasing concentration of selumetinib in cells with different mutational status. Microarray results: C, Venn-diagram summarizing differentially expressed genes in selumetinib-treated GNAQ^Q209L/P cell lines (green circle), BRAF^V600E (red circle) and WT cells (blue circle), with corresponding overlapping genes as indicated. D, Heat map representation of top 19 genes identified as meeting the statistical threshold (see methods) for significant change in expression in all the cell lines and with fold-change > 2 in GNAQ^Q209L/P cell lines, in response to 250nM selumetinib (grey bar), or DMSO as control for 8 hours, in triplicate. One replicate of the Mel290 cell line was excluded as it did not cluster with the other two replicates. Cell lines with individual replicates are columns, genes are rows.

Figure 2. Validation of MEK-regulated genes in GNAQ^Q209L/P cells

These genes were representative of those shared between GNAQ^Q209L/P and BRAF^V600E MEK-dependent signature. A, Immunoblot analysis of cells treated with 250nM selumetinib over the time, using antibodies for the indicated proteins. Each blot is representative of at least two experiments showing same results. B, C, D, Relative mRNA levels of selected ERK output genes before and after selumetinib in three GNAQ^Q209L/P cell lines. Cells were treated with selumetinib for 8h and RT-PCR was performed using gene-specific primers for DUSP6, ETV5, SPRY2 and cyclin D1. Values were normalized with GADPH and HPRT as housekeeping genes using the ΔΔCT method. Each experiment was performed in triplicates. Bars, ± sd.

Figure 3. Confirmation of MEK-dependent genes differentially regulated by selumetinib in GNAQ^Q209L/P cells

A, Immunoblot analysis of DDX21, CDK5R1 and JUN expression after treatment with 250nM selumetinib over the time in cell lines with different mutational status. Total RNA was extracted from cells after 8h of treatment and qRT-PCR was performed using gene-specific primers for DDX21 (B), CDK5R1 (C) and JUN (D). Values are relative to mRNA levels of untreated cells. Each experiment was performed two or three times in triplicates. E, Western blot analysis of UM cells after GNAQ knockdown by siRNA using the indicated antibodies.

Figure 4. GNAQ^Q209L/P-MEK-dependent genes play a role in cell proliferation and metastasis

A, siRNA mediated knockdown of DDX21 (+) in five cell lines (lower panel); (-), control siRNA. Cell viability was measured after 4 days (upper panel). B, Migration assays of WT and GNAQ^Q209L/P cell lines transfected with CDK5R1 or control siRNA (lower panels). Cells migrated through the membrane pores were stained and counted under the microscope. Representative fields of x200 magnification are shown. C, UM cell lines were trasnfected with control or c-Jun siRNA and tested in proliferation assays (D) after 4 days of 250nM selumetinib treatment. Experiments were repeated three times in triplicates. * p<0.0001; ** p<0.001, *** p<0.005 for comparison of c-Jun versus control siRNA with selumetinib treatment.

Figure 5. Validation of MEK inhibition and expression of ERK-dependent genes in tumor tissues

A, Baseline and day 14 liver tumor biopsies were collected in patients with mutant GNAQ/11 metastatic uveal melanoma receiving selumetinib in a phase II clinical trial. Matched-pair biopsies were analyzed by immunoblotting for expression levels of pERK, ERK, Cyclin D1, CDK5R1, DDX21, c-Jun and α-tubulin. Clinical efficacy is also shown: PR= partial response, SD= stable disease, PD= progressive disease. B, Liver metastases of patient A with uveal melanoma (pre- and post-treatment with selumetinib). A partial response in the liver lesion is assessed by CAT scan (left, arrows) and PET scan (right, arrows).