RSK regulates activated BRAF signalling to mTORC1 and promotes melanoma growth

Abstract

The Ras/mitogen-activated protein kinase (MAPK) signalling cascade regulates various biological functions, including cell growth, proliferation and survival. As such, this pathway is often deregulated in cancer, including melanomas, which frequently harbour activating mutations in the NRAS and BRAF oncogenes. Hyperactive MAPK signalling is known to promote protein synthesis, but the mechanisms by which this occurs remain poorly understood. Here, we show that expression of oncogenic forms of Ras and Raf promotes the constitutive activation of the mammalian target of rapamycin (mTOR). Using pharmacological inhibitors and RNA interference, we find that the MAPK-activated protein kinase RSK (p90 ribosomal S6 kinase) is partly required for these effects. Using melanoma cell lines carrying activating BRAF mutations, we show that ERK/RSK signalling regulates assembly of the translation initiation complex and polysome formation, as well as the translation of growth-related messenger RNAs containing a 5'-terminal oligopyrimidine (TOP) motif. Accordingly, we find that RSK inhibition abrogates tumour growth in mice. Our findings indicate that RSK may be a valuable therapeutic target for the treatment of tumours characterized by deregulated MAPK signalling, such as melanoma.

Fig. 1

Oncogenic RAS and RAF promote mTORC1 signalling in a RSK-dependent manner. (A) HEK293 cells stably expressing constitutively-activated MEK1 (MEK-DD), Ras (G12V) or Raf (V600E) were serum-starved overnight, and analyzed for ERK and RSK phosphorylation by immunoblotting. Immunoprecipitated RSK1 kinase activity was assayed using GST-rpS6 as substrate, in the presence of γ[³²P]ATP. Samples were subjected to SDS-PAGE and the dried coomassie-stained gel autoradiographed. (B) HEK293 cells stably expressing constitutively-activated MEK1, Ras or Raf were serum-starved overnight, pre-treated with U0126 (U0), PD184352 (PD), SL0101 (SL), BI-D1870 (BI) or rapamycin (RAP) for 1h, prior to being harvested. Immunoprecipitated S6K1 kinase activity was assayed using GST-rpS6 as substrate, in the presence of γ[³²P]ATP. The histogram shows quantifications of phosphorylated rpS6 from three independent experiments. Phosphorylation of endogenous rpS6 and S6K1, and total rpS6 protein level were monitored by immunoblotting. (C) Control and constitutively-activated cells were maintained in low-serum (2%) for 18 hr. Global protein synthesis was measured by adding 0.5 μCi/ml [³H]leucine to the medium for 6 hours. Histograms show radioactivity incorporation normalized to time 0. (D) As in (C), except that cells were treated with indicated inhibitors for 60 minutes prior to addition of 0.5 μCi/ml [³H]leucine to the medium.

Fig. 2

Inhibition of ERK/RSK signalling decreases constitutive activation of mTORC1 in melanoma. (A) Four melanoma cell lines were used in this study. While A375 and Colo829 cells harbour a B-Raf V600E mutation, WM852 and WM1361 cells carry an N-Ras mutation at Q61 (R or K). (B) Phosphorylation of endogenous RSK, ERK1/2, rpS6, S6K1 and 4E-BP1 was monitored in total extracts from serum-starved melanoma cell lines and normal human melanocytes treated or not with insulin (100 nM) for 30 min. Cell lysates were also immunoblotted for total protein levels (RSK1, ERK1, rpS6, S6K1, 4E-BP1 and β-actin). (C) Serum-starved melanoma cells were treated with the indicated inhibitors for 60 minutes. Immunoprecipitated S6K1 kinase activity was assayed as in Fig. 1. (D) Phosphorylation of endogenous rpS6 and S6K1, and total rpS6 protein level were monitored by immunoblotting.

Fig. 3

RSK contributes to the constitutive activation of mTORC1 in melanoma. (A) Relative mRNA expression of the RSK isoforms in Colo829, A375, WM1361 and WM852 melanoma cell lines. Histograms show relative abundance of all RSK mRNAs monitored by quantitative real-time PCR and normalized to control HPRT1 mRNA abundance. (B) A375 and WM1361 cells stably expressing a control shRNA or shRNAs against RSK1/2 were serum-starved for 18 hours. Phosphorylation of endogenous rpS6 and S6K1, and total rpS6, RSK1, RSK2 and β-actin protein level were monitored by immunoblotting.

Fig. 4

ERK/RSK signalling promotes mTORC1-mediated eIF4F assembly and translation in melanoma. (A) Serum-starved melanoma cells were treated with PD184352, BI-D1870, KU-0063794 or rapamycin (50 nM) for 60 minutes. Time course analysis of [³H]leucine incorporation was performed as in Fig. 1, except that cells were collected at 0, 2, 4 and 6 h after addition of [³H]leucine. (B) Serum-starved Colo829 cells were treated with indicated inhibitors as in (A). Cell extracts were size-fractionated by centrifugation through sucrose gradients (20-50%). The absorbance of polysomes (P) and subpolysomal (S) particles was continuously monitored at 260 nm. Representative A₂₆₀ nm traces are shown (n = 3). The area under the curves was calculated and the P/S ratio refers to the percentage of ribosomes engaged in translation. The data are normalized to P/S ratio of control condition (DMSO) and presented as a mean ± S.E. (n = 3). (C) Association of 4E-BP1 and eIF4G to the 7-methylguanosine cap complex was monitored by immunoblotting 7-methylguanosine precipitates from serum-starved Colo829 cells, pre-treated with the indicated inhibitors for 60 minutes. Equal levels between precipitates were monitored through analysis of eIF4E. Phosphorylation and total protein level of endogenous 4E-BP1 and ERK1/2 were monitored by immunoblotting.

Fig. 5

ERK/RSK signalling promotes translation of growth-related mRNAs in melanoma. (A) Scatter plot of mRNAs log2-coefficients in polysomal mRNA from serum-starved Colo829 cells treated with mTOR (rapamycin or KU-0063794) or MAPK (BI-D1870 or PD184352) pathway inhibitors. Red and grey lines represent cutoffs of 1.5-fold (log2-coefficient=−0.58) and 1.25-fold (log2-coefficient=−0.32) decrease, respectively. mRNAs regulated by both types of inhibitors are found in the bottom-left quadrant delimited by two red lines whereas mRNAs regulated by only one inhibitor class are found at the top-left (MAPK) or bottom-right of the plot (mTOR). (B) Gene categories found as enriched by DAVID analysis in the list of downregulated mRNAs identified as translationally repressed by both mTOR and MAPK inhibitors (treatment effect<1.5-fold decrease and pvalue <0.05). (C) K-Means clustering of downregulated mRNAs in the presence of both mTOR and MAPK inhibitors led to the identification of a cluster whose mRNAs are translationally repressed. mRNAs and sample class are presented vertically and horizontally, respectively. Green and red represent respectively a relative decrease and a relative increase in mRNA abundance compared to the mRNA mean abundance. (D) Profile of a selected mRNA identified by clustering with log2 signal on the y-axis and the different sample classes on the x-axis. (E) Enrichment analysis using Ingenuity Pathways Analysis of mRNAs from identified clusters.

Fig. 6

RSK is essential for melanoma cell proliferation and tumour development in mice. (A) A375 and Colo829 cells stably expressing control vector or shRNA against RSK1/2 were grown in culture medium containing 5% FBS. The relative number of viable cells was measured during four days using an MTS assay. The data are normalized to control vector (Ctl). (B) As in (A), except that after 72 h of culture, the relative number of viable cells was measured by cell counts. (C) Graph showing the relative number of apoptotic cells after 48 h of culture in serum-growing conditions, as measured by FACS analysis of Annexin V binding. (D) As in (A), except that A375 cells were grown in the presence of indicated inhibitors. (E) As in (C), except that A375 cells were treated with the indicated inhibitor for 24 h prior to FACS analysis. As positive control, cells were treated with 0.5 μM doxorubicin (DOX). (F) and (G) A375 and Colo829 cells stably expressing a control shRNA or shRNAs against RSK1/2 were injected subcutaneously into the flanks of athymic mice. Mice were monitored for tumour development and graphs represent the growth rate of subcutaneous tumours. Values represent the average volume +/− SEM of 3 to 5 tumours (3 to 5 mice). Insets: Endogenous RSK1, RSK2 and β-actin protein levels in injected cells were monitored by immunoblotting.