Writers, readers and erasers of RNA modifications in cancer

Although cancer was originally considered a disease driven only by genetic mutations, it has now been proven that it is also an epigenetic disease driven by DNA hypermethylation-associated silencing of tumor suppressor genes and aberrant histone modifications. Very recently, a third component has emerged: the so-called epitranscriptome understood as the chemical modifications of RNA that regulate and alter the activity of RNA molecules. In this regard, the study of genetic and epigenetic disruption of the RNA-modifying proteins is gaining momentum in advancing our understanding of cancer biology. Furthermore, the development of epitranscriptomic anticancer drugs could lead to new promising and unexpected therapeutic strategies for oncology in the coming years

The lincRNA MIRAT binds to IQGAP1 and modulates the MAPK pathway in NRAS mutant melanoma.

Despite major advances in targeted melanoma therapies, drug resistance limits their efficacy. Long noncoding RNAs (lncRNAs) are transcriptome elements that do not encode proteins but are important regulatory molecules. LncRNAs have been implicated in cancer development and response to different therapeutics and are thus potential treatment targets; however, the majority of their functions and molecular interactions remain unexplored. In this study, we identify a novel cytoplasmic intergenic lincRNA (MIRAT), which is upregulated following prolonged MAPK inhibition in NRAS mutant melanoma and modulates MAPK signaling by binding to the MEK scaffold protein IQGAP1. Collectively, our results present MIRAT's direct modulatory effect on the MAPK pathway and highlight the relevance of cytoplasmic lncRNAs as potential targets in drug resistant cancer

BRAF activation by metabolic stress promotes glycolysis sensitizing NRASQ61-mutated melanomas to targeted therapy

Glycolysis; Melanomas; Targeted therapyGlucólisis; Melanomas; Terapia dirigidaGlucòlisi; Melanomes; Teràpia dirigidaNRAS-mutated melanoma lacks a specific line of treatment. Metabolic reprogramming is considered a novel target to control cancer; however, NRAS-oncogene contribution to this cancer hallmark is mostly unknown. Here, we show that NRASQ61-mutated melanomas specific metabolic settings mediate cell sensitivity to sorafenib upon metabolic stress. Mechanistically, these cells are dependent on glucose metabolism, in which glucose deprivation promotes a switch from CRAF to BRAF signaling. This scenario contributes to cell survival and sustains glucose metabolism through BRAF-mediated phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2/3 (PFKFB2/PFKFB3). In turn, this favors the allosteric activation of phosphofructokinase-1 (PFK1), generating a feedback loop that couples glycolytic flux and the RAS signaling pathway. An in vivo treatment of NRASQ61 mutant melanomas, including patient-derived xenografts, with 2-deoxy-D-glucose (2-DG) and sorafenib effectively inhibits tumor growth. Thus, we provide evidence for NRAS-oncogene contributions to metabolic rewiring and a proof-of-principle for the treatment of NRASQ61-mutated melanoma combining metabolic stress (glycolysis inhibitors) and previously approved drugs, such as sorafenib.This work was funded by Instituto de Salud Carlos III and co-funded by European Union (ERDF/ESF, “A way to make Europe”/“Investing in your future”) PI14/0375-Fondos FEDER J.A.R., PI17/00043-Fondos FEDER; J.A.R., PI20/0384-Fondos FEDER; J.A.R., Euronanomed2-ISCIII (AC16/00019)-Fondos FEDER; J.A.R., Asociación Española Contra el Cancer (AECC-GCB15152978SOEN) (supported P.G.M., K.M.); J.A.R., Ramón Areces Foundation (supported K.M. and research); J.A.R. (PI17/00412)-Fondos FEDER; R.B., A.M., A.N.S. We thank A. Zorzano’s laboratory for technical assistance and performance of Seahorse technology

BRAF activation by metabolic stress promotes glycolysis sensitizing NRASQ61-mutated melanomas to targeted therapy

NRAS-mutated melanoma lacks a specific line of treatment. Metabolic reprogramming is considered a novel target to control cancer; however, NRAS-oncogene contribution to this cancer hallmark is mostly unknown. Here, we show that NRAS(Q61)-mutated melanomas specific metabolic settings mediate cell sensitivity to sorafenib upon metabolic stress. Mechanistically, these cells are dependent on glucose metabolism, in which glucose deprivation promotes a switch from CRAF to BRAF signaling. This scenario contributes to cell survival and sustains glucose metabolism through BRAF-mediated phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2/3 (PFKFB2/PFKFB3). In turn, this favors the allosteric activation of phosphofructokinase-1 (PFK1), generating a feedback loop that couples glycolytic flux and the RAS signaling pathway. An in vivo treatment of NRAS(Q61) mutant melanomas, including patient-derived xenografts, with 2-deoxy-D-glucose (2-DG) and sorafenib effectively inhibits tumor growth. Thus, we provide evidence for NRAS-oncogene contributions to metabolic rewiring and a proof-of-principle for the treatment of NRAS(Q61)-mutated melanoma combining metabolic stress (glycolysis inhibitors) and previously approved drugs, such as sorafenib. Targeted therapeutic options for NRAS-mutant melanoma are limited. Here, the authors show that under metabolic stress NRAS-mutant melanoma cells activate a BRAF-dependent glycolysis pathway for survival, leading to improve efficacy of sorafenib when combined with glycolysis inhibitors

Uncoupling of the LKB1-AMPKα Energy Sensor Pathway by Growth Factors and Oncogenic BRAFV600E

BACKGROUND: Understanding the biochemical mechanisms contributing to melanoma development and progression is critical for therapeutical intervention. LKB1 is a multi-task Ser/Thr kinase that phosphorylates AMPK controlling cell growth and apoptosis under metabolic stress conditions. Additionally, LKB1(Ser428) becomes phosphorylated in a RAS-Erk1/2-p90(RSK) pathway dependent manner. However, the connection between the RAS pathway and LKB1 is mostly unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using the UV induced HGF transgenic mouse melanoma model to investigate the interplay among HGF signaling, RAS pathway and PI3K pathway in melanoma, we identified LKB1 as a protein directly modified by HGF induced signaling. A variety of molecular techniques and tissue culture revealed that LKB1(Ser428) (Ser431 in the mouse) is constitutively phosphorylated in BRAF(V600E) mutant melanoma cell lines and spontaneous mouse tumors with high RAS pathway activity. Interestingly, BRAF(V600E) mutant melanoma cells showed a very limited response to metabolic stress mediated by the LKB1-AMPK-mTOR pathway. Here we show for the first time that RAS pathway activation including BRAF(V600E) mutation promotes the uncoupling of AMPK from LKB1 by a mechanism that appears to be independent of LKB1(Ser428) phosphorylation. Notably, the inhibition of the RAS pathway in BRAF(V600E) mutant melanoma cells recovered the complex formation and rescued the LKB1-AMPKalpha metabolic stress-induced response, increasing apoptosis in cooperation with the pro-apoptotic proteins Bad and Bim, and the down-regulation of Mcl-1. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that growth factor treatment and in particular oncogenic BRAF(V600E) induces the uncoupling of LKB1-AMPKalpha complexes providing at the same time a possible mechanism in cell proliferation that engages cell growth and cell division in response to mitogenic stimuli and resistance to low energy conditions in tumor cells. Importantly, this mechanism reveals a new level for therapeutical intervention particularly relevant in tumors harboring a deregulated RAS-Erk1/2 pathway

HGF induces LKB1^Ser431 phosphorylation in a RAS-p90^RSK dependent manner.

<p>(A) Five µg of phospho-protein isolated complexes from samples: untreated (Control), HGF triggered (40 ng/ml) w/o PHA (0,2 µM) were resolved by SDS-PAGE. p-LKB1^Ser428, p-Erk1/2^{Thr202/Tyr204} and Erk2 antibodies were probed against the membrane. Ponceau S staining of membrane is showed for phospho-protein extracts loading control. (B) 37-31E, 37-31T, SKMel28, and MeWo cells were treated in serum starvation conditions with HGF (40 ng/ml), U0126 (10 µM) and LY294002 (10 µM) as indicated in the figure. Western-blots show the levels of the indicated proteins. (C) Time course showing the phosphorylation of the LKB1^Ser431 and p-90^RSK^{Thr359/Ser363} after HGF triggering (40 ng/ml) under serum starvation conditions. LKB1 total protein is shown as a loading control. On the right, LKB1^Ser431 is phosphorylated in response to HGF in an Erk1/2-p90^RSK dependent manner. Time course shows the phosphorylation of Erk1/2^{Thr202/Tyr204} and LKB1^Ser428 in B16F1 cells. Down below, 37-31E melanoma cells were serum starved and triggered with HGF (40 ng/ml) for 5 minutes. Then, cells were treated with the Mek1/2 specific inhibitor U0126 (10 µM) for the indicated increasing times. Fifty µg of total lysates were resolved by SDS-PAGE and membrane was probed with the indicated antibodies. (D) 37-31E cells were treated for 10 min in serum starvation with HGF (40 ng/ml) in the presence or absence of U0126 (10 µM) or BI-D1870 (10 µM). Western-blots show the levels of p-LKB1^Ser431, p-Erk1/2^{Thr202/Tyr204} and p-CREB^Ser133.</p

Inhibition of oncogenic BRAF^V600E signaling restores the limited response to metabolic stress of BRAF mutant melanoma cell lines.

<p>(A) BRAF mutant melanoma cells have a limited response to energy withdrawal that is restored by U0126 treatment. Fifty micrograms of total lysates from UACC903, A375, SKMel28 and 37-31E melanoma cells grown in serum free high glucose medium (H.G.), serum free low glucose medium (L.G.) or serum free low glucose medium (L.G.) plus 10 µM of U0126 for 4 hours were separated by SDS-PAGE. Western-Blot shows the activation status of proteins in the RAS and LKB1-AMPK-mTOR pathways. (B) U0126 inhibitor treatment does not activate AMPK. UACC903 A375 and SKMel28 melanoma cells were grown in high glucose medium with serum in the absence or presence of 1 µM, 5 µM or 10 µM of U0126. Total protein lysates were subjected to SDS-PAGE. Western-blot shows the phosphorylation state of AMPKα in the presence of different concentrations of U0126. (C) Inhibition of BRAF signaling increases cell response to AICAR. UACC903 and SKMel28 cells were grown in complete medium; cells were treated with AICAR (1 mM) for 4 h in the presence or absence of U0126 (10 µM) inhibitor. p-LKB1^Ser428, p-AMPK^Thr172, p-Erk1/2^{Thr202/Tyr204}, p-ACC^Ser79 levels were checked by western blot. (D) Sorafenib treatment and siRNA BRAF knockdown restores the metabolic stress pathway in BRAF mutant melanoma cells. In the left panel, UACC903, A375 and SKMel28 melanoma cells were grown in low glucose serum free medium+/−EGF (50 ng/ml) for 4 h in the presence or absence of U0126 (10 µM) Western blots show the levels of p-AMPKα^T172, p-LKB1^Ser431 p-Erk1/2^{Thr202/Tyr204} and pCREB^Ser133 proteins under the different conditions. In the right panel SKMel28 cells were transfected with either a scramble siRNA or BRAF siRNA. 72 hours after transfection, cells were starved either in high glucose (H.G.) or low glucose (L.G.) medium for six hours. Western-blots show the levels of p-AMPKα^T172, p-Erk1/2^{Thr202/Tyr204} and BRAF proteins. (E) p90^Rsk inhibitor BI-D1870 (10 µM), does not restore the metabolic stress pathway. UACC903, A375 and SKMel28 melanoma cells were grown in low glucose serum free medium+/−EGF (50 ng/ml) for 4 h in the presence or absence of BI-D1870 (10 µM). Western blots show the levels of the indicated proteins under the different conditions.</p

Restoration of the LKB1-AMPKα pathway in BRAF^V600E melanoma cells induces apoptosis under energy stress conditions.

<p>(A) UACC903, A375 and SKMel28 human melanoma cells were grown in complete medium (H.G.), or low glucose medium (L.G.) with or without 10 µM of the Mek1/2 specific inhibitor U0126 for 12 h. Then, Annexin V and PI (propidium iodide) positive cells were analyzed by flow cytometry. Histograms show the result from FACS analysis. Graphs on the right show the percentage of viable and dead cells in a parallel experiment under the same conditions determined by nuclear staining exclusion (Guava-ViaCount). (B) Time course at 4 and 12 hours showing the LKB1-AMPKα pathway status under the same conditions. UACC903, A375 and SKMel28 human melanoma cells were grown in complete medium (high glucose H.G.), low glucose medium (L.G.) with or without 10 µM of the Mek1/2 specific inhibitor U0126 for the times indicated. Fifty micrograms of total protein lysates were separated by SDS-PAGE and same membranes were blotted against the indicated antibodies. All experiments were done at least three times. Representative experiments are shown. (C) UACC903 cells were transfected either with a scramble siRNA or with equimolar amounts of AMPKα1 and AMPKα2 siRNAs for a total concentration of 100 nM. 72 hours after transfection cells were starved in low glucose medium for 6 hours in the presence or absence of 10 µM of U0126. Dead cells were quantified by nuclear staining exclusion (Guava-ViaCount). Western-blots show the levels of p-AMPKα^T172, AMPKα and p-Erk1/2^{Thr202/Tyr204} under the different conditions. (D) UACC903 and A375 melanoma cells were grown in complete medium (H.G. cm), serum free high glucose medium (H.G. sf), serum free low glucose medium (L.G.), serum free complete medium plus U0126 10 µM (H.G.+U0126) and low glucose serum free medium plus U0126 10 µM (L.G.+U0126) for 12 hours. The levels of Bim, phospho-Bad and Mcl-1 are showed under the different experimental conditions.</p

LKB1^Ser431 (Ser428 human) is phosphorylated in response to different growth factors, in BRAF^V600E mutant melanoma cells and mouse tumor samples.

<p>(A) B16F1, 37-31T and MeWo cells were serum starved and treated with HGF (40 ng/ml), EGF (100 ng/ml), FGF2 (100 ng/ml), Herregulin (50 ng/ml), IGF-1 (50 ng/ml), PDGF (50 ng/ml), TNF-α (100 ng/ml) Insulin (100 nM) and TPA (200 nM). Fifty µg of total lysates were separated by SDS-PAGE and same membranes were incubated against the indicated antibodies. (B) MeWo (BRAF wild type), A375 (BRAF^V600E), SKMel28 (BRAF^V600E) and UACC903 (BRAF^V600E) human melanoma cells were growth in complete medium (CM) or serum starvation (SF) conditions as indicated. Fifty µg of total lysates were analyzed by SDS-PAGE. The phosphorylation status of LKB1^Ser428, p-Erk1/2^{Thr202/Tyr204} and p-90^{RSK Thr359/Ser363} is shown. Total Erk1/2 is used as a loading control. Cell genotypes are showed. (C) p-LKB1^Ser431 and p-Erk1/2^{Thr202/Tyr204} levels in mouse melanoma tumor samples. Samples 1–7 primary tumors raised in HGF-UV irradiated transgenic mice. Samples 8 and 9 show xenograph tumors generated from 37-31E cells in FVB mice with high and low p-Erk1/2 levels, respectively. As a control fifty micrograms of protein from 37-31E melanoma cell line treated with HGF (40 ng/ml) for 10 minutes was added (Total lysates, T.L.). Same membrane was blotted against the indicated antibodies. Quantifications of phospho-proteins normalized against total protein are showed in the graphs below.</p

Dual MEK/AKT inhibition with trametinib and GSK2141795 does not yield clinical benefit in metastatic NRAS-mutant and wild-type melanoma.

Aberrant MAPK and PI3K pathway signaling may drive the malignant phenotype in NRAS-mutant and BRAFWT NRASWT metastatic melanoma. To target these pathways, NRAS-mutant and BRAFWT NRASWT patients received oral trametinib at 1.5&nbsp;mg daily and GSK2141795 at 50&nbsp;mg daily in a two-cohort Simon two-stage design. Participants had adequate end-organ function and no more than two prior treatment regimens. Imaging assessments were performed at 8-week intervals. A total of 10 NRAS-mutant and 10 BRAFWT NRASWT patients were enrolled. No objective responses were noted in either cohort. The median PFS and OS were 2.3 and 4.0&nbsp;months in the NRAS-mutant cohort and 2.8 and 3.5&nbsp;months in the wild-type cohort. Grade 3 and grade 4 adverse events, primarily rash, were observed in 25% of patients. We conclude that the combination of trametinib and GSK2141795 does not have significant clinical activity in NRAS-mutant or BRAFWT NRASWT melanoma