The majority of uveal melanomas carry oncogenic mutations in the G proteins GNAQ and GNA11, with consequent activation of the MAPK pathway. Selective MEK inhibitors, such as selumetinib, have shown clinical benefit in uveal melanoma. However, mechanisms of drug resistance limit their efficacy in some patients. Analysis of MEK inhibitor–resistant uveal melanoma cell lines revealed the induction of RAS protein expression and activity. This effect was mediated by the RNA helicase DDX43, which was remarkably overexpressed in these cells. Depletion of DDX43 in MEK inhibitor–resistant cells decreased RAS proteins and inhibited ERK and AKT pathways. On the contrary, ectopic expression of DDX43 in parental uveal melanoma cells induced RAS protein levels and rendered cells resistant to MEK inhibition. Similar to DDX43 depletion, downregulation of KRAS, HRAS, and NRAS inhibited downstream pathways in the resistant cells, overcoming mutant GNAQ signaling. We also analyzed the expression of DDX43 in liver metastases of patients with uveal melanoma by RT-PCR, and found a significant overexpression of DDX43 in patients who did not benefit from selumetinib therapy. In conclusion, DDX43 induces RAS protein expression and signaling, mediating a novel mechanism of MEK inhibitor resistance. The detection of DDX43 in patients with uveal melanoma could lead to more targeted therapies for this disease. Mol Cancer Ther; 13(8); 2073–80. ©2014 AACR.

Uveal melanoma accounts for approximately 5% of all melanomas. Most arise from melanocytes within the uveal tract, which consists of the iris, ciliary body, and choroid of the eye (1). This type of melanoma has a very high tendency to metastasize to the liver (2). As opposed to cutaneus melanoma, BRAF and RAS mutations are absent or extremely rare in uveal melanoma (3). Rather, activating mutations in G-protein α-subunits, GNAQ or GNA11, have been detected in nearly 80% of primary uveal melanomas (4–6), and mediate the activation of the MAPK pathway (4). The prognosis of patients with metastatic uveal melanoma is poor with a median 1-year survival rate of less than 30% (7, 8). Nearly all patients with uveal melanoma die due to metastatic disease and there are limited therapeutic options (9, 10). We have reported that the small-molecule MEK inhibitor, selumetinib, can inhibit pERK and cyclin D1, resulting in decreased viability of uveal melanoma cell lines (11). Furthermore, in patients with uveal melanoma, selumetinib treatment can result in tumor shrinkage, and the sustained inhibition of pERK may be predictive of benefit (12). Recently, our group reported the results of a randomized phase II clinical trial of selumetinib versus temozolomide in patients with metastatic uveal melanoma (13). These results indicated an improvement in the response rate and a significant median progression-free survival. Thus, selumetinib is the first drug in medical oncology to ever show a clinical benefit in patients with this disease. Despite these promising results, “de novo” and “acquired” resistance to this drug and other MEK inhibitors remains a major clinical problem (14). For example, resistance to selumetinib has been described in colorectal cancer cells carrying BRAF and RAS mutations, where resistance is mediated by the amplification of the driving oncogene (15, 16). KRAS overexpression has also been implicated in resistance to MEK inhibition in colon carcinoma (17). RAS activation has been described as a mechanism of acquired resistance to BRAF inhibitors in cutaneus melanoma with BRAF mutation, due to receptor tyrosine kinase (RTK) activation (18) or NF1 loss (19). In uveal melanoma with GNAQ/GNA11 mutations, the mechanisms of resistance to MEK inhibitors are unknown and understanding this process will prove critical to develop more effective therapies for the treatment of this disease.

DDX43, also called HAGE, is a member of the DEAD-box family of ATP-dependent RNA helicases. It was first identified as a cancer/testis antigen, and it is highly expressed in many tumor types compared with normal tissues (20). In particular, DDX43 is overexpressed in 50% of acute myeloid leukemias (21), and its expression was associated with advanced disease and poor prognosis (22). These helicases browse RNA molecules and promote the dissociation of the RNA from ribonucleoproteins (23). In this way, they support processes such as gene transcription and posttranscriptional regulation, including pre-mRNA splicing, translation initiation/elongation, and RNA degradation (24–26). Their altered expression levels have been also implicated in tumor initiation, progression, and maintenance (27). Recently, DDX43 was found highly expressed in melanoma (28), and it promoted protein expression of NRAS by unwinding of mRNA secondary structures, and enhancing NRAS translation (29). Here, we report that the expression of DDX43 is elevated in uveal melanoma cells with acquired resistance to selumetinib and in patients with metastatic uveal melanoma who did not benefit from selumetinib treatment. Furthermore, we demonstrated that DDX43 regulates RAS protein expression and AKT activation. Selumetinib-resistant cells became sensitive to AKT inhibition, suggesting alternative strategies for the treatment of patients with uveal melanoma with high DDX43 expression and resistance to MEK inhibitors.

Cell lines and reagents

The GNAQ (Q209P)-mutant cell lines, Omm1.3 and Mel270, were kindly provided by Dr. Bruce Ksander (Harvard Medical School, Boston, MA) in 2009. The cells have been sequenced for the presence of mutations in codons 209 (exon 5) and 183 (exon 4) of GNAQ and GNA11. A karyotype test was also performed for the cell line in 2012, confirming the authenticity of the cell lines as reported previously (30). The resistant cell lines, Rs-Omm13 and Rs-Mel270, were derived from the corresponding cell lines after at least 3 weeks of exposure to escalating doses up to 1 μmol/L of selumetinib. Cells were cultured in RPMI medium supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, and maintained at 37°C in 5% CO2. Cells were treated with selumetinib (AZD6244, ARRY-142866, AstraZeneca) and MK2206 (supplied by Merck; ref. 31), PD0325901 (32), and GSK1120212 (Selleck Chemicals; ref. 33).

Cell viability assays

Cells were plated in 96-well plates, and treated with the indicated concentrations of drugs. Viability was assessed after 4 days of treatment using the Cell Counting Kit 8 from Dojindo Molecular Technologies according to the manufacturer's instructions. Cell viability is expressed as a percentage of untreated cells.

Immunoblotting and RAS-GTP pull-down assays

The cells and the biopsy tissues were lysed in RIPA buffer supplemented with protease inhibitor cocktail tablets (Roche Diagnostics) and 1 mmol/L NaVO3. Total protein concentration of the lysates was measured by BCA assay (Bio-Rad), and equal amounts of protein were loaded on 4% to 12% PAGE gels (Invitrogen). Polyvinylidene difluoride membranes were blocked with 5% nonfat dried milk in PBS buffer containing 0.1% Tween-20 (PBST) and probed with antibody for pERK, ERK, pAKT, pan-AKT, pRB, c-Jun, cyclin D1, tubulin (Cell Signaling Technology), DDX43 (Abcam), KRAS, HRAS (Abnova), and NRAS (Santa Cruz Biotechnology). GTP-bound RAS was measured using the RAF RAS-binding domain (RBD) pull-down and Detection Kit (Thermo Scientific), as instructed by the manufacturers.

RNAi-mediated gene knockdown

DDX43-1 and -2 siRNA were from OriGene. siRNA against GNAQ (sc-35429), KRAS (sc-35731), NRAS (sc-36004), HRAS (sc-29340), and control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology. They were transfected using Lipofectamine RNAiMAX reagent (Life Technologies). The human DDX43 cDNA clone in pCMV6-XL5 was obtained from OriGene.

Tissue sample collection and qRT-PCR

Matched tumor biopsies were collected from patients with metastatic uveal melanoma (clinicaltrials.gov, # NCT01143402). The protocol was approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center (New York, NY), and all patients signed informed consent forms. Total RNA was extracted from flash-frozen specimens and cell lines using TRIzol (Life Technologies) followed by DNase digestion and RNeasy (Qiagen) column purification following the manufacturer's protocol. The RNA integrity was verified using an Agilent Bioanalyzer 2100 (Agilent). Of note, 1 μg of total RNA was reverse transcribed using the Thermoscript RT-PCR system (Life Technologies). The resultant cDNA was used in RT-PCR reactions using an iCycler (Bio-Rad) with predesigned TaqMan Gene expression Assays for DDX43 and GAPDH genes (Life Technologies). Triplicate Ct values were averaged, the targets were interpolated from the standard curves and normalized to GAPDH. The results are shown as DDX43/GAPDH ratio and analyzed with the Wilcoxon Mann–Whitney test for two-sided P value.

GNAQ-mutant cell lines with acquired resistance to selumetinib display differential expression of RAS and DDX43 and deregulated MAPK/AKT pathways

To explore mechanisms of drug resistance, we generated MEK inhibitor–resistant uveal melanoma cell lines. The GNAQ-mutant cell lines, Omm1.3 and Mel270, which are sensitive to selumetinib treatment (34), were chronically exposed to increasing doses of selumetinib. The resulting cell lines, Rs-Omm1.3 and Rs-Mel270, showed that IC50 were at least 10-fold higher than their parental cells, Omm1.3 and Mel270, in cell viability assays (Fig. 1A). The cells also showed cross resistance to the MEK inhibitors PD0325901 and GSK1120212 (Supplementary Fig. S1). We then examined the expression levels and phosphorylation status of different components of the ERK pathway after MEK inhibition. In parental cells Omm1.3 and Mel270, increasing doses of selumetinib for 24 hours caused a decrease in pERK, cyclin D1, and pRb (Fig. 1B), whereas c-Jun and pAKT were slightly increased. In Rs-Omm1.3 and Rs-Mel270, pERK was also inhibited by the treatments (Fig. 1B), even though the signal seems to rebound over time (Supplementary Fig. S2). Furthermore, the downstream signaling was deregulated in these cells, with sustained expression of cyclin D1, pRb, and high expression of c-Jun and pAKT, independently of treatments (Fig. 1B).

Figure 1.

Selumetinib-resistant cells exhibit RAS and DDX43 upregulation and increased ERK/AKT pathway activation. A, after continuous exposure to selumetinib, the cell lines Rs-Omm1.3 and Rs-Mel270 became resistant to the drug compared with their parental cells, Omm1.3 and Mel270. Cell viability on day 4 was calculated as percentage of untreated controls. Each point is a mean ± SD. B, immunoblot analysis of parental and selumetinib-resistant Omm1.3 (left) and Mel270 (right) cells treated with increasing concentrations of selumetinib for 24 hours. Both selumetinib-resistant cell lines Rs-Omm1.3 and Rs-Mel270 showed sustained expression of cyclin D1, pRB, c-Jun, pAKT, and RAS independently of treatment. C, whole-cell lysates were subjected to pull-down assays with GST-bound CRAF RAS-binding domain (RBD) for active RAS. The cells were treated with selumetinib for 24 hours or treated and washed afterwards and kept in culture for 24 hours (ND, no drug). The active RAS pull-down (top bands) and total lysates (bottom bands) were immunoblotted with a pan-RAS antibody. D, parental and selumetinib-resistant cells were analyzed for DDX43 expression by immunoblotting, before and after selumetinib treatments. E, real-time PCR was performed using gene-specific primers and the values were normalized to GAPDH as housekeeping gene. Relative mRNA levels of DDX43 in parental and selumetinib-resistant cell lines are shown. Each experiment was performed in triplicate. Bars, ± SD.

Figure 1.

Selumetinib-resistant cells exhibit RAS and DDX43 upregulation and increased ERK/AKT pathway activation. A, after continuous exposure to selumetinib, the cell lines Rs-Omm1.3 and Rs-Mel270 became resistant to the drug compared with their parental cells, Omm1.3 and Mel270. Cell viability on day 4 was calculated as percentage of untreated controls. Each point is a mean ± SD. B, immunoblot analysis of parental and selumetinib-resistant Omm1.3 (left) and Mel270 (right) cells treated with increasing concentrations of selumetinib for 24 hours. Both selumetinib-resistant cell lines Rs-Omm1.3 and Rs-Mel270 showed sustained expression of cyclin D1, pRB, c-Jun, pAKT, and RAS independently of treatment. C, whole-cell lysates were subjected to pull-down assays with GST-bound CRAF RAS-binding domain (RBD) for active RAS. The cells were treated with selumetinib for 24 hours or treated and washed afterwards and kept in culture for 24 hours (ND, no drug). The active RAS pull-down (top bands) and total lysates (bottom bands) were immunoblotted with a pan-RAS antibody. D, parental and selumetinib-resistant cells were analyzed for DDX43 expression by immunoblotting, before and after selumetinib treatments. E, real-time PCR was performed using gene-specific primers and the values were normalized to GAPDH as housekeeping gene. Relative mRNA levels of DDX43 in parental and selumetinib-resistant cell lines are shown. Each experiment was performed in triplicate. Bars, ± SD.

Close modal

We next analyzed the expression of RAS in all the cell lines. The parental cells expressed low levels of RAS, which slightly increased with higher doses of selumetinib. In contrast, RAS protein levels were elevated in the resistant cells regardless of treatments (Fig. 1B).

To test whether RAS protein expression corresponded to increased activity, we used RAF-RBD pull-down assays. In the parental Omm1.3 and Mel270 cells, basal RAS expression and activity were relatively low and temporarily induced by selumetinib without changes in RAS expression (Fig. 1C). This activation of RAS is possibly mediated by the downregulation of the Sprouty proteins by ERK inhibition, as reported (35). We have also shown that GNAQ-mutant cells have elevated expression of Spry2 that is downregulated by selumetinib (34). In contrast, both RAS expression and activity were elevated in Rs-Omm1.3 and Rs-Mel270 cell lines, and remained active even when selumetinib was removed for 24 hours (Fig. 1C). These results show that the selumetinib-resistant cells have sustained activation of the RAS, ERK, and AKT pathways.

To determine whether the increase of RAS protein corresponded to higher RAS mRNA levels, we performed RT-PCR from parental and MEK inhibitor–resistant cells with or without treatment. We did not find any difference in RNA expression for KRAS or NRAS (data not shown). Rs-Omm1.3 and Rs-Mel270 cells were sequenced for MEK, KRAS, NRAS, HRAS mutations and these were negative. FISH analysis was performed to exclude gene amplification of the oncogene GNAQ or RAS proteins. Assays to detect activation of RTK were also negative.

It has been reported that the DEAD-box RNA helicase antigen DDX43 regulates NRAS protein expression through unwinding of duplex RNA in melanoma cells (29). Thus, we analyzed the expression of DDX43 in uveal melanoma cells, and found a remarkable upregulation of DDX43 in the MEK inhibitor–resistant cells, compared with low or no expression in their parental cells (Fig. 1D). Treatments with selumetinib did not further increase DDX43 expression either with increasing doses (Fig. 1D) or over time (Supplementary Fig. S2). The mRNA expression of DDX43 was analyzed by RT-PCR in all the cell lines, showing a 9-fold and 7.6-fold increase in Rs-Omm1.3 and Rs-Mel270 cells, compared with the respective parental cell lines (Fig. 1E).

DDX43 mediates the expression of RAS and resistance to selumetinib

To determine whether DDX43 was involved in MEK inhibitor resistance, DDX43 was overexpressed in the selumetinib-sensitive uveal melanoma parental cell lines, Omm1.3 and Mel270. In these cells, DDX43 induced the protein levels of KRAS, HRAS, NRAS, and increased pAKT (Fig. 2A). Surprisingly, the levels of pERK did not change, possibly because the pathway is regulated by mutant GNAQ through PKC activation, as reported (36). Nevertheless, higher expression of RAS has important implications as it regulates multiple pathways, including the PI3K/AKT pathway (37, 38). Next, we tested whether DDX43 affected the response to selumetinib. DDX43-expressing uveal melanoma cells were more resistant to selumetinib treatment compared with cells transfected with an empty vector (V) in viability assays (Fig. 2B), suggesting that DDX43 may mediate mechanisms of resistance to MEK inhibition through RAS and AKT activation.

Figure 2.

DDX43 mediates the expression of RAS and resistance to selumetinib. A, parental uveal melanoma (UM) cell lines Omm1.3 and Mel270 were transfected with DDX43 cDNA or an empty vector (V). Cell lysates were subjected to Western blot analysis for expression of DDX43, KRAS, HRAS, NRAS, pERK, and pAKT. B, the cell line Omm1.3 and Mel270 overexpressing DDX43 are less sensitive to increasing doses of selumetinib, after 4 days of treatments. Graph reports the mean ± SD of three independent experiments. C, DDX43 downregulation decreases RAS protein expression/activity and restores MEK sensitivity. siRNA-mediated knockdown of DDX43 (+) and control siRNA (−) in the MEK inhibitor–resistant cell lines decreases KRAS, HRAS, NRAS and downstream signaling molecules pERK, pAKT, and c-Jun. D, pull-down assays for RAS activity in cells after DDX43 downregulation. Total RAS, DDX43 levels, and tubulin are also shown. E, cell viability of Rs-Omm1.3 and Rs-Mel270 cells was measured after DDX43 knockdown and 3 days of selumetinib treatments. Bars are the mean of three experiments, expressed as percentage of untreated cells, ± SD. *, P < 0.001 and #, P < 0.002 for comparison of DDX43 siRNA with and without selumetinib treatment.

Figure 2.

DDX43 mediates the expression of RAS and resistance to selumetinib. A, parental uveal melanoma (UM) cell lines Omm1.3 and Mel270 were transfected with DDX43 cDNA or an empty vector (V). Cell lysates were subjected to Western blot analysis for expression of DDX43, KRAS, HRAS, NRAS, pERK, and pAKT. B, the cell line Omm1.3 and Mel270 overexpressing DDX43 are less sensitive to increasing doses of selumetinib, after 4 days of treatments. Graph reports the mean ± SD of three independent experiments. C, DDX43 downregulation decreases RAS protein expression/activity and restores MEK sensitivity. siRNA-mediated knockdown of DDX43 (+) and control siRNA (−) in the MEK inhibitor–resistant cell lines decreases KRAS, HRAS, NRAS and downstream signaling molecules pERK, pAKT, and c-Jun. D, pull-down assays for RAS activity in cells after DDX43 downregulation. Total RAS, DDX43 levels, and tubulin are also shown. E, cell viability of Rs-Omm1.3 and Rs-Mel270 cells was measured after DDX43 knockdown and 3 days of selumetinib treatments. Bars are the mean of three experiments, expressed as percentage of untreated cells, ± SD. *, P < 0.001 and #, P < 0.002 for comparison of DDX43 siRNA with and without selumetinib treatment.

Close modal

We also evaluated the effect of DDX43 depletion in the MEK inhibitor–resistant cells by using two siRNA sequences (siDDX43-1, Fig. 2C; and siDDX43-2, Supplementary Fig. S3). Silencing of DDX43 corresponded to decreased protein expression of KRAS, NRAS, HRAS and the downstream effectors pERK, pAKT, and c-Jun in both resistant cell lines (Fig. 2C). DDX43 depletion did not change KRAS or NRAS mRNA levels (Supplementary Fig. S4) as previously reported (29), suggesting that DDX43 regulates RAS posttranscriptionally. The decrease in RAS protein levels induced by DDX43 depletion corresponded to a reduction in RAS activity in both MEK inhibitor–resistant cell lines (Fig. 2D). Most importantly, the depletion of DDX43 caused inhibition of cell viability of the MEK inhibitor–resistant cells, and this effect was further enhanced by the treatment with selumetinib (Fig. 2E), suggesting that DDX43 is required for MEK inhibitor resistance.

To confirm that the effects of DDX43 depletion are mediated by RAS downregulation, each RAS protein was silenced by gene-specific siRNA. KRAS and HRAS knockdown caused downregulation of pMEK, pERK, and pAKT in both cell lines (Fig. 3A), whereas NRAS did not. c-Jun was downregulated by KRAS and NRAS, whereas HRAS regulated expression of c-Jun only in Rs-Mel270. We have reported that GNAQ silencing inhibits pERK and pAKT, and decreases cell viability of the parental GNAQ-mutant uveal melanoma cells (31). We also tested the contribution of mutant GNAQ on downstream signaling pathways in the resistant cells. GNAQ downregulation induced minimal effects on downstream pathways (Fig. 3B). Similar to DDX43 downregulation, KRAS and HRAS depletion decreased cell viability of the selumetinib-resistant cell lines, whereas NRAS had partial effects (Fig. 3C). GNAQ depletion did not affect cell viability of Rs-Omm1.3 and Rs-Mel270 cells (Fig. 3C), further demonstrating that RAS proteins become the dominant mediators of cell signaling in these cells.

Figure 3.

RAS proteins regulate cell signaling in MEK inhibitor–resistant cells. A, selumetinib-resistant Rs-Omm1.3 (left) and Rs-Mel270 (right) cells were transfected with siRNA for KRAS, HRAS, and NRAS and analyzed for downstream signaling, that is, pMEK, pERK, pAKT, and c-Jun by immunoblotting. B, the cells were also transfected with GNAQ siRNA and analyzed for downstream signaling. C, KRAS and HRAS are necessary for survival of MEK inhibitor–resistant uveal melanoma (UM) cells. Rs-Omm1.3 and Rs-Mel270 cells depleted of each RAS protein or GNAQ were tested in cell viability assays after 72 hours of siRNA transfection. *, P < 0.0001 and **, P < 0.001 for comparison of cells transfected with siKRAS and siHRAS versus control siRNA, for both cell lines.

Figure 3.

RAS proteins regulate cell signaling in MEK inhibitor–resistant cells. A, selumetinib-resistant Rs-Omm1.3 (left) and Rs-Mel270 (right) cells were transfected with siRNA for KRAS, HRAS, and NRAS and analyzed for downstream signaling, that is, pMEK, pERK, pAKT, and c-Jun by immunoblotting. B, the cells were also transfected with GNAQ siRNA and analyzed for downstream signaling. C, KRAS and HRAS are necessary for survival of MEK inhibitor–resistant uveal melanoma (UM) cells. Rs-Omm1.3 and Rs-Mel270 cells depleted of each RAS protein or GNAQ were tested in cell viability assays after 72 hours of siRNA transfection. *, P < 0.0001 and **, P < 0.001 for comparison of cells transfected with siKRAS and siHRAS versus control siRNA, for both cell lines.

Close modal

AKT inhibition inhibits cell viability of selumetinib-resistant GNAQ-mutant cells

Elevated DDX43 and RAS expression mediated the activation of downstream survival pathways, especially pAKT (Figs. 1B and 2A). Thus, we assessed the effects of AKT inhibition in uveal melanoma cell lines. The AKT inhibitor MK2206 (AKTi) inhibited AKT phosphorylation in parental and MEK inhibitor–resistant cells (Fig. 4A). In cell proliferation assays, AKT inhibition had minimal effects in the parental cells, Omm1.3 and Mel270 (Fig. 4B), while it greatly inhibited cell viability of the resistant cell lines, Rs-Omm1.3 and Rs-Mel270 (Fig. 4B), suggesting that in these cells the DDX43/RAS-mediated activation of AKT is necessary for cell survival, and its inhibition may be a means of overcoming MEK inhibitor resistance.

Figure 4.

Inhibition of AKT decreases cell viability of MEK inhibitor–resistant cells. A, parental and selumetinib-resistant uveal melanoma (UM) cells were treated with 1.5 μmol/L of MK2206 (AKTi) for 24 hours and analyzed by immunoblotting for target inhibition of pAKT. B, parental and resistant cells were exposed to increasing concentrations of AKTi (0–5 μmol/L) and analyzed in viability assays after 4 days of treatment. Graph shows the mean of three independent experiments ± SD.

Figure 4.

Inhibition of AKT decreases cell viability of MEK inhibitor–resistant cells. A, parental and selumetinib-resistant uveal melanoma (UM) cells were treated with 1.5 μmol/L of MK2206 (AKTi) for 24 hours and analyzed by immunoblotting for target inhibition of pAKT. B, parental and resistant cells were exposed to increasing concentrations of AKTi (0–5 μmol/L) and analyzed in viability assays after 4 days of treatment. Graph shows the mean of three independent experiments ± SD.

Close modal

DDX43 is highly expressed in liver metastasis of patients with uveal melanoma

To determine the relevance of DDX43 expression in patients with uveal melanoma, we assessed the expression of DDX43 by RT-PCR in liver metastasis of patients with uveal melanoma carrying GNAQ or GNA11 mutations who were treated on the phase II study of selumetinib. We analyzed biopsies from 14 patients, 6 of whom had partial response or stable disease for ≥16 weeks, defined as “responders” (R) according to RECIST, and 8 patients who had disease progression before 16 weeks with no evidence of radiological response, defined as “nonresponders” (NR). The “responder” patients had low expression of DDX43 (Fig. 5A). In contrast, DDX43 was highly expressed in the “nonresponder” group, and there was a statistically significant association with poor outcome (P = 0.045). The tissues from six representative patients were also analyzed by immunoblotting for protein expression. The patients who responded to therapy had complete inhibition of pERK with selumetinib treatment, whereas the “nonresponders” showed none or minimal inhibition (Fig. 5B). The protein levels of DDX43 in biopsy tissues were low in “responders,” whereas it was highly expressed in “nonresponders.” Similarly, levels of total KRAS and NRAS were elevated in the “nonresponder” group, while HRAS was not detectable in these specimens.

Figure 5.

DDX43 mRNA and protein levels are higher in liver biopsies of “responder” versus “nonresponder” patients. A, DDX43 expression was analyzed by RT-PCR in biopsies of 14 patients (6 “responders” and 8 “nonresponders”). Triplicate values were normalized with GAPDH as housekeeping gene, and reported as a box plot showing significant association with poor outcome in patients with uveal melanoma treated with selumetinib. B, liver biopsies from six representative patients (P) before (−) and after (+) selumetinib treatment were analyzed by immunoblotting with antibodies for pERK, DDX43, KRAS, NRAS, and tubulin.

Figure 5.

DDX43 mRNA and protein levels are higher in liver biopsies of “responder” versus “nonresponder” patients. A, DDX43 expression was analyzed by RT-PCR in biopsies of 14 patients (6 “responders” and 8 “nonresponders”). Triplicate values were normalized with GAPDH as housekeeping gene, and reported as a box plot showing significant association with poor outcome in patients with uveal melanoma treated with selumetinib. B, liver biopsies from six representative patients (P) before (−) and after (+) selumetinib treatment were analyzed by immunoblotting with antibodies for pERK, DDX43, KRAS, NRAS, and tubulin.

Close modal

The RAS/RAF/MEK/ERK pathway is often activated by genetic alterations in upstream signaling molecules, such as RAS and BRAF in cutaneous melanoma, and GNAQ/11 in uveal melanoma (4, 5). Hyperactivation of this signaling cascade results in dysregulated cell proliferation and malignant transformation. Targeting this pathway with MEK inhibitors has shown activity in different types of cancers (39, 40), including uveal melanoma (13). However, resistance to MEK inhibitors is common and undermines the efficacy of these treatments (41, 42). Resistance to MEK inhibitors has been extensively described in cutaneus melanoma with BRAF and RAS mutations (43). Also in colon carcinoma, MEK inhibition leads to gene amplification of RAS and activation of downstream pathways (16). Activated RAS has been linked to MEK inhibitor resistance as it drives the activation of both the PI3K-AKT and MEK-ERK pathways (37). Here, we found that in uveal melanoma cell lines, the sustained inhibition of MEK leads to increased expression of DDX43, and consequent induction of RAS and downstream pathways. Furthermore, using RT-PCR, we found that DDX43 is overexpressed in patients who did not respond to selumetinib treatment, confirming the in vitro findings. Altered expression of DDX43 has been reported in human cancers (27, 44). In particular, DDX43 was required for cell proliferation of malignant melanoma-initiating cells by promoting the expression of NRAS through RNA unwinding (29). However, this is the first time that DDX43 has been linked to MEK inhibitor resistance through the regulation of RAS. Interestingly, it was reported that MEK inhibitor–resistant colon carcinoma cells showed increased KRAS expression, which represented a potential mechanism of MEK inhibitor resistance (17). However, the modalities of KRAS protein induction remained to be established.

Recently, Lito and colleagues have reported that RAF inhibitors cause relief of negative feedback with activation of RAS and rebound of ERK activity, without changes in RAS expression (35). We have shown a similar activation of RAS, which was induced by MEK inhibition in the parental cells. However, this effect was sustained in uveal melanoma selumetinib-resistant cells, where the expression of both DDX43 and RAS was elevated. DDX43 silencing decreased RAS proteins and downstream signaling, i.e., pERK and pAKT, making DDX43 a mediator of MEK resistance that could represent a class effect to all MEK inhibitors. Little and colleagues reported an increase in c-Jun in colorectal cancer cells with acquired resistance to selumetinib (16). Indeed, c-Jun was highly expressed in MEK inhibitor–resistant cells, and it could be downregulated by knockdown of DDX43 and RAS, but not GNAQ, further confirming the importance of RAS signaling in these cells.

In our cells, DDX43 mediated “acquired” resistance to the MEK inhibitor, as its expression increased after long-term treatments. However, DDX43 seemed to mediate intrinsic resistance to selumetinib in patients with uveal melanoma with elevated expression at baseline. It was reported that DDX43 is highly expressed in chronic myeloid leukemia due to gene demethylation (22), and correlated with poorer prognosis in terms of cytogenetic response to drug treatments. Similar changes in DDX43 gene methylation may occur in uveal melanoma.

DDX43 has been also identified as a tumor-specific gene expressed in sarcoma (20), and it has been associated with poor clinical outcome in patients with breast cancer (45). Recently, several studies have provided new insights on RNA helicases and their emerging roles in cell signaling. For example, DDX3, another member of this family, was found to regulate the Wnt–β-catenin network in a genome-wide siRNA screen in human embryonic kidney cells (46). Another report showed DDX5 as a regulator of alternative splicing of HRAS (47).

Our results provide evidence that DDX43 increased the expression and activation of RAS in uveal melanoma cells with GNAQ mutation, and mediated resistance to MEK inhibitors.

Finally, these findings would indicate that DDX43 may have predictive value in determining who will most benefit from a MEK inhibitor therapy in this disease.

R.D. Carvajal is a consultant/advisory board member for AstraZeneca. No potential conflicts of interest were disclosed by the other authors.

Conception and design: G. Ambrosini, R.D. Carvajal, G.K. Schwartz

Development of methodology: G. Ambrosini, G.K. Schwartz

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.D. Carvajal, G.K. Schwartz

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): G. Ambrosini, R. Khanin, R.D. Carvajal, G.K. Schwartz

Writing, review, and/or revision of the manuscript: G. Ambrosini, R.D. Carvajal, G.K. Schwartz

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): G. Ambrosini, R.D. Carvajal, G.K. Schwartz

Study supervision: G. Ambrosini, R.D. Carvajal, G.K. Schwartz

G. Ambrosini, Cycle for Survival (Philanthropy).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Strickland
D
,
Lee
JA
. 
Melanomas of eye: stability of rates
.
Am J Epidemiol
1981
;
113
:
700
02
.
2.
Singh
AD
,
Bergman
L
,
Seregard
S
. 
Uveal melanoma: epidemiologic aspects
.
Ophthalmol Clin North Am
2005
;
18
:
75
84
.
3.
Zuidervaart
W
,
van Nieuwpoort
F
,
Stark
M
,
Dijkman
R
,
Packer
L
,
Borgstein
AM
, et al
Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS
.
British J Cancer
2005
;
92
:
2032
8
.
4.
Van Raamsdonk
CD
,
Bezrookove
V
,
Green
G
,
Bauer
J
,
Gaugler
L
,
O'Brien
JM
, et al
Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi
.
Nature
2009
;
457
:
599
602
.
5.
Van Raamsdonk
CD
,
Griewank
KG
,
Crosby
MB
,
Garrido
MC
,
Vemula
S
,
Wiesner
T
, et al
Mutations in GNA11 in uveal melanoma
.
The New England J Med
2010
;
363
:
2191
9
.
6.
Onken
MD
,
Worley
LA
,
Long
MD
,
Duan
S
,
Council
ML
,
Bowcock
AM
, et al
Oncogenic mutations in GNAQ occur early in uveal melanoma
.
Investig Ophthalmol Visual Sci
2008
;
49
:
5230
4
.
7.
Gragoudas
ES
,
Egan
KM
,
Seddon
JM
,
Glynn
RJ
,
Walsh
SM
,
Finn
SM
, et al
Survival of patients with metastases from uveal melanoma
.
Ophthalmology
1991
;
98
:
383
9
;.
8.
Kath
R
,
Hayungs
J
,
Bornfeld
N
,
Sauerwein
W
,
Hoffken
K
,
Seeber
S
. 
Prognosis and treatment of disseminated uveal melanoma
.
Cancer
1993
;
72
:
2219
23
.
9.
Patel
M
,
Smyth
E
,
Chapman
PB
,
Wolchok
JD
,
Schwartz
GK
,
Abramson
DH
, et al
Therapeutic implications of the emerging molecular biology of uveal melanoma
.
Clin Cancer Res
2011
;
17
:
2087
100
.
10.
Yonekawa
Y
,
Kim
IK
. 
Epidemiology and management of uveal melanoma
.
Hemat/Oncol Clin North Am
2012
;
26
:
1169
84
.
11.
Ambrosini
G
,
Pratilas
CA
,
Qin
LX
,
Tadi
M
,
Surriga
O
,
Carvajal
RD
, et al
Identification of unique MEK-dependent genes in GNAQ mutant uveal melanoma involved in cell growth, tumor cell invasion, and MEK-resistance
.
Clin Cancer Res
2012
;
18
:
1
10
.
12.
Carvajal
RD AG
,
Wolchok
JD
,
Chapman
PB
,
Dickson
MA
,
D'Angelo
SP
, et al
Pharmacodynamic activity of selumetinib to predict radiographic response in advanced uveal melanoma
.
J Clin Oncol
2012
;
30
(
suppl; abstr 8598
).
13.
Carvajal
RD SJ
,
Quevedo
F
,
Milhem
MM
,
Joshua
AM
,
Kudchadkar
RR
, et al
Phase II study of selumetinib (sel) versus temozolomide (TMZ) in gnaq/Gna11 (Gq/11) mutant (mut) uveal melanoma (UM)
.
J Clin Oncol
2013
;
31
(
suppl; abstr CRA9003
).
14.
Little
AS
,
Balmanno
K
,
Sale
MJ
,
Smith
PD
,
Cook
SJ
. 
Tumour cell responses to MEK1/2 inhibitors: acquired resistance and pathway remodelling
.
Biochem Society Trans
2012
;
40
:
73
8
.
15.
Corcoran
RB
,
Dias-Santagata
D
,
Bergethon
K
,
Iafrate
AJ
,
Settleman
J
,
Engelman
JA
. 
BRAF gene amplification can promote acquired resistance to MEK inhibitors in cancer cells harboring the BRAF V600E mutation
.
Science Signal
2010
;
3
:
ra84
.
16.
Little
AS
,
Balmanno
K
,
Sale
MJ
,
Newman
S
,
Dry
JR
,
Hampson
M
, et al
Amplification of the driving oncogene, KRAS or BRAF, underpins acquired resistance to MEK1/2 inhibitors in colorectal cancer cells
.
Science Signal
2011
;
4
:
ra17
.
17.
Wang
Y
,
Van Becelaere
K
,
Jiang
P
,
Przybranowski
S
,
Omer
C
,
Sebolt-Leopold
J
. 
A role for K-ras in conferring resistance to the MEK inhibitor, CI-1040
.
Neoplasia
2005
;
7
:
336
47
.
18.
Nazarian
R
,
Shi
H
,
Wang
Q
,
Kong
X
,
Koya
RC
,
Lee
H
, et al
Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation
.
Nature
2010
;
468
:
973
7
.
19.
Whittaker
SR
,
Theurillat
JP
,
Van Allen
E
,
Wagle
N
,
Hsiao
J
,
Cowley
GS
, et al
A genome-scale RNA interference screen implicates NF1 loss in resistance to RAF inhibition
.
Cancer Dis
2013
;
3
:
350
62
.
20.
Martelange
V
,
De Smet
C
,
De Plaen
E
,
Lurquin
C
,
Boon
T
. 
Identification on a human sarcoma of two new genes with tumor-specific expression
.
Cancer Res
2000
;
60
:
3848
55
.
21.
Adams
SP
,
Sahota
SS
,
Mijovic
A
,
Czepulkowski
B
,
Padua
RA
,
Mufti
GJ
, et al
Frequent expression of HAGE in presentation chronic myeloid leukaemias
.
Leukemia
2002
;
16
:
2238
42
.
22.
Roman-Gomez
J
,
Jimenez-Velasco
A
,
Agirre
X
,
Castillejo
JA
,
Navarro
G
,
San Jose-Eneriz
E
, et al
Epigenetic regulation of human cancer/testis antigen gene, HAGE, in chronic myeloid leukemia
.
Haematologica
2007
;
92
:
153
62
.
23.
Fuller-Pace
FV
. 
DExD/H box RNA helicases: multifunctional proteins with important roles in transcriptional regulation
.
Nucleic Acids Res
2006
;
34
:
4206
15
.
24.
Glisovic
T
,
Bachorik
JL
,
Yong
J
,
Dreyfuss
G
. 
RNA-binding proteins and post-transcriptional gene regulation
.
FEBS Lett
2008
;
582
:
1977
86
.
25.
Linder
P
,
Jankowsky
E
. 
From unwinding to clamping - the DEAD box RNA helicase family
.
Nature Rev Molec Cell Biol
2011
;
12
:
505
16
.
26.
Jarmoskaite
I
,
Russell
R
. 
DEAD-box proteins as RNA helicases and chaperones
.
WIREs RNA
2011
;
2
:
135
52
.
27.
Robert
F
,
Pelletier
J
. 
Perturbations of RNA helicases in cancer
.
WIREs RNA
2013
;
4
:
333
49
.
28.
Mathieu
MG
,
Knights
AJ
,
Pawelec
G
,
Riley
CL
,
Wernet
D
,
Lemonnier
FA
, et al
HAGE, a cancer/testis antigen with potential for melanoma immunotherapy: identification of several MHC class I/II HAGE-derived immunogenic peptides
.
Cancer Immunol Immunother
2007
;
56
:
1885
95
.
29.
Linley
AJ
,
Mathieu
MG
,
Miles
AK
,
Rees
RC
,
McArdle
SE
,
Regad
T
. 
The helicase HAGE expressed by malignant melanoma-initiating cells is required for tumor cell proliferation in vivo
.
J Biol Chem
2012
;
287
:
13633
43
.
30.
Griewank
KG
,
Yu
X
,
Khalili
J
,
Sozen
MM
,
Stempke-Hale
K
,
Bernatchez
C
, et al
Genetic and molecular characterization of uveal melanoma cell lines
.
Pigment Cell Melanoma Res
2012
;
25
:
182
7
.
31.
Ambrosini
G
,
Musi
E
,
Ho
AL
,
de Stanchina
E
,
Schwartz
GK
. 
Inhibition of mutant GNAQ signaling in uveal melanoma induces AMPK-dependent autophagic cell death
.
Mol Cancer Ther
2013
;
12
:
768
76
.
32.
Thompson
N
,
Lyons
J
. 
Recent progress in targeting the Raf/MEK/ERK pathway with inhibitors in cancer drug discovery
.
Curr Opinion Pharmacol
2005
;
5
:
350
6
.
33.
Gilmartin
AG
,
Bleam
MR
,
Groy
A
,
Moss
KG
,
Minthorn
EA
,
Kulkarni
SG
, et al
GSK1120212 (JTP-74057) is an inhibitor of MEK activity and activation with favorable pharmacokinetic properties for sustained in vivo pathway inhibition
.
Clin Cancer Res
2011
;
17
:
989
1000
.
34.
Ambrosini
G
,
Pratilas
CA
,
Qin
LX
,
Tadi
M
,
Surriga
O
,
Carvajal
RD
, et al
Identification of unique MEK-dependent genes in GNAQ mutant uveal melanoma involved in cell growth, tumor cell invasion, and MEK resistance
.
Clin Cancer Res
2012
;
18
:
3552
61
.
35.
Lito
P
,
Pratilas
CA
,
Joseph
EW
,
Tadi
M
,
Halilovic
E
,
Zubrowski
M
, et al
Relief of profound feedback inhibition of mitogenic signaling by RAF inhibitors attenuates their activity in BRAFV600E melanomas
.
Cancer Cell
2012
;
22
:
668
82
.
36.
Chen
X
,
Wu
Q
,
Tan
L
,
Porter
D
,
Jager
MJ
,
Emery
C
, et al
Combined PKC and MEK inhibition in uveal melanoma with GNAQ and GNA11 mutations
.
Oncogene
2013 Oct 21
.
[Epub ahead of print]
.
37.
Karnoub
AE
,
Weinberg
RA
. 
Ras oncogenes: split personalities
.
Nature Rev Mol Cell Biol
2008
;
9
:
517
31
.
38.
Rodriguez-Viciana
P
,
Warne
PH
,
Dhand
R
,
Vanhaesebroeck
B
,
Gout
I
,
Fry
MJ
, et al
Phosphatidylinositol-3-OH kinase as a direct target of Ras
.
Nature
1994
;
370
:
527
32
.
39.
Neuzillet
C
,
Tijeras-Raballand
A
,
de Mestier
L
,
Cros
J
,
Faivre
S
,
Raymond
E
. 
MEK in cancer and cancer therapy
.
Pharmacol Ther
2014
;
141
:
160
71
.
40.
Friday
BB
,
Adjei
AA
. 
Advances in targeting the Ras/Raf/MEK/Erk mitogen-activated protein kinase cascade with MEK inhibitors for cancer therapy
.
Clin Cancer Res
2008
;
14
:
342
6
.
41.
Little
AS
,
Smith
PD
,
Cook
SJ
. 
Mechanisms of acquired resistance to ERK1/2 pathway inhibitors
.
Oncogene
2013
;
32
:
1207
15
.
42.
Poulikakos
PI
,
Solit
DB
. 
Resistance to MEK inhibitors: should we co-target upstream?
Sci Signal
2011
;
4
:
pe16
.
43.
Das Thakur
M
,
Stuart
DD
. 
The evolution of melanoma resistance reveals therapeutic opportunities
.
Cancer Res
2013
;
73
:
6106
10
.
44.
Abdelhaleem
M
. 
Over-expression of RNA helicases in cancer
.
Anticancer Res
2004
;
24
:
3951
3
.
45.
Chan
S. A-FTMA
,
McArdle
S
,
Moseley
P
,
Johnson
C
,
Andrew
R
. 
HAGE (DDX43) protein expression as an independent biomarker of poor clinical outcome of breast cancer (BC) and potential as a therapeutic target for ER-negative BC
.
J Clinical Oncol
2012
;
30
(
suppl; abstr 1013
).
46.
Cruciat
CM
,
Dolde
C
,
de Groot
RE
,
Ohkawara
B
,
Reinhard
C
,
Korswagen
HC
, et al
RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-beta-catenin signaling
.
Science
2013
;
339
:
1436
41
.
47.
Camats
M
,
Guil
S
,
Kokolo
M
,
Bach-Elias
M
. 
P68 RNA helicase (DDX5) alters activity of cis- and trans-acting factors of the alternative splicing of H-Ras
.
PloS ONE
2008
;
3
:
e2926
.