Journal: Oncogene

Article Title: RSK regulates activated BRAF signalling to mTORC1 and promotes melanoma growth

doi: 10.1038/onc.2012.312

Figure Lengend Snippet: 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.

Article Snippet: Cell Culture and treatments The cell lines used in this study were purchased from ATCC (A375, Colo829 and SK-MEL-2) or Coriell (WM1361 and WM852).

Techniques: Inhibition, Activation Assay, Mutagenesis, Phospho-proteomics, Immunoprecipitation, Activity Assay, Western Blot

Journal: Oncogene

Article Title: RSK regulates activated BRAF signalling to mTORC1 and promotes melanoma growth

doi: 10.1038/onc.2012.312

Figure Lengend Snippet: 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.

Article Snippet: Cell Culture and treatments The cell lines used in this study were purchased from ATCC (A375, Colo829 and SK-MEL-2) or Coriell (WM1361 and WM852).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Stable Transfection, shRNA, Phospho-proteomics, Western Blot

Journal: Oncogene

Article Title: RSK regulates activated BRAF signalling to mTORC1 and promotes melanoma growth

doi: 10.1038/onc.2012.312

Figure Lengend Snippet: 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.

Article Snippet: Cell Culture and treatments The cell lines used in this study were purchased from ATCC (A375, Colo829 and SK-MEL-2) or Coriell (WM1361 and WM852).

Techniques: Centrifugation, Control, Western Blot, Phospho-proteomics

Journal: Oncogene

Article Title: RSK regulates activated BRAF signalling to mTORC1 and promotes melanoma growth

doi: 10.1038/onc.2012.312

Figure Lengend Snippet: 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.

Article Snippet: Cell Culture and treatments The cell lines used in this study were purchased from ATCC (A375, Colo829 and SK-MEL-2) or Coriell (WM1361 and WM852).

Techniques:

Journal: Oncogene

Article Title: RSK regulates activated BRAF signalling to mTORC1 and promotes melanoma growth

doi: 10.1038/onc.2012.312

Figure Lengend Snippet: 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.

Article Snippet: Cell Culture and treatments The cell lines used in this study were purchased from ATCC (A375, Colo829 and SK-MEL-2) or Coriell (WM1361 and WM852).

Techniques: Stable Transfection, Expressing, Control, Plasmid Preparation, shRNA, MTS Assay, Binding Assay, Positive Control, Injection, Western Blot