Supplementary Figure 2 from Vemurafenib Resistance Signature by Proteome Analysis Offers New Strategies and Rational Therapeutic Concepts
Verena PaulitschkeWalter BergerPhilipp PaulitschkeElisabeth HofstätterBernhard KnappRuth Dingelmaier‐HovorkaDagmar FödingerWalter JägerThomas SzekeresAnastasia MeshcheryakovaAndrea BileckChristine PirkerHubert PehambergerChristopher GernerRainer Kunstfeld
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<p>Proteome characterization of Vemurafenib-sensitive and-resistant melanoma cells V0 and V1 of nuclear fraction. Left: Hierarchical clustering of Z-scored expression values for significantly changed proteins, revealing differences between V0 and V1. Left: Profiles of the five main clusters. Two clusters with the highest difference are selected and the significant regulated groups are depicted. Red: upregulated in V1, green: downregulated in V1.</p>Keywords:
Proteome
Hierarchical clustering
Signature (topology)
BRAF Inhibitors and Radiation Do Not Act Synergistically to Inhibit WT and V600E BRAF Human Melanoma
Recent evidence suggests that melanoma patients treated with BRAF inhibitors experience radiosensitization with an increased frequency of side-effects. This could also imply increased effectiveness when treating melanoma.To test whether the BRAF inhibitors dabrafenib and vemurafenib together with ionizing radiation more effectively inhibit melanoma cells, primary human melanoma tumor cell lines expressing wild-type (WT) or mutant V600E BRAF were analyzed by cell survival, cell death, and cell-cycle testing.All melanoma cell lines examined were radioresistant in these assays. BRAF inhibitor treatment alone suppressed cell survival more effectively than radiation in all the mutant V600E BRAF cell lines, and vemurafenib, but not dabrafenib, also inhibited cell survival in the WT BRAF cell lines at clinically relevant concentrations. However, when cells were treated with BRAF inhibitor followed by radiation, there was no increased effect on the suppression of cell survival. Vemurafenib induced more necrosis than radiation in most melanoma cell lines, irrespective of BRAF status, but this effect was not additive with the combination treatment. BRAF inhibitors and radiation had variable, but independent effects on the induction of cell-cycle arrest.These results suggest that BRAF inhibitors and ionizing radiation do not act synergistically to inhibit the growth of primary human melanoma cells.
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Tumor heterogeneity affects the efficacy of anticancer treatment as tumor subclones with distinct molecular patterns may be present within one tumor, leading to differing sensitivities to chemotherapeutic agents. In the present study, six melanoma tissue fragments were obtained from different parts of tumor of four patients and then the effect of vemurafenib treatment on biological characteristics and molecular processes of cell cultures was estimated by using MTT-test, apoptosis, migration and invasion assays, PCR real time. There was different BRAF status determined between cells derived from the central and peripheral regions of primary melanoma tumors. BRAF-positive melanoma cells showed an increased apoptotic rate under vemurafenib treatment, as well as increased migration and invasion rates, whereas BRAF-negative melanoma cells did not exhibit such tendency. Furthermore, semaphorin-5A levels were diminished in BRAF-positive cells, but not in BRAF-negative ones, which could be related to increased migration and invasion. Melanoma cells derived from different regions of the same tumor may differ by mutations status, molecular processes and biological response to target therapy. The downregulation of semaphorin-5A may be involved in divergent effects of anticancer agents on tumor cell biology.
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BRAF inhibitors are commonly used in targeted therapies for melanoma patients harboring BRAFV600E mutant. Despite the benefit of vemurafenib therapy, acquired resistance during or after treatment remains a major obstacle in BRAFV600E mutant melanoma. Here we found that RSK2 is overexpressed in melanoma cells and the high expression of RSK2 indicates poor overall survival (OS) in melanoma patients. Overexpression of RSK2 leads to vemurafenib resistance, and the deletion of RSK2 inhibits cell proliferation and sensitizes melanoma cells to vemurafenib. Mechanistically, RSK2 enhances the phosphorylation of FOXO1 by interacting with FOXO1 and promoting its subsequent degradation, leading to upregulation of cyclin D1 in melanoma cells. These results not only reveal the presence of a RSK2-FOXO1-cyclin D1 signaling pathway in melanoma, but also provide a potential therapeutic strategy to enhance the efficacy of vemurafenib against cancer.
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Abstract Melanoma is molecularly and structurally heterogeneous, with some tumor cells existing under hypoxic conditions. Our cell growth assays showed that under controlled hypoxic conditions, BRAF(V600E) melanoma cells rapidly became resistant to vemurafenib. By employing both a three-dimensional (3D) spheroid model and a two-dimensional (2D) hypoxic culture system to model hypoxia in vivo, we identified upregulation of HGF/MET signaling as a major mechanism associated with vemurafenib resistance as compared with 2D standard tissue culture in ambient air. We further confirmed that the upregulation of HGF/MET signaling was evident in drug-resistant melanoma patient tissues and mouse xenografts. Pharmacologic inhibition of the c-Met/Akt pathway restored the sensitivity of melanoma spheroids or 2D hypoxic cultures to vemurafenib. Mol Cancer Ther; 15(10); 2442–54. ©2016 AACR.
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Purpose: Many patients with BRAFV600E mutant melanoma treated with BRAF inhibitors experience a rapid response, but ultimately develop resistance. Insight into the mechanism of resistance is critical for development of more effective treatment strategies.Experimental Design: Comprehensive genomic profiling of serial biopsies was performed in a patient with a BRAFV600E mutant metastatic melanoma who developed resistance to vemurafenib. An AGAP3-BRAF fusion gene, identified in the vemurafenib-resistant tumor, was expressed in BRAFV600E melanoma cell lines, and its effect on drug sensitivity was evaluated.Results: Clinical resistance to vemurafenib in a melanoma harboring a BRAFV600E mutation was associated with acquisition of an AGAP3-BRAF fusion gene. Expression of the AGAP3-BRAF fusion in BRAFV600E mutant melanoma cells induced vemurafenib resistance; however, these cells remained relatively sensitive to MEK inhibitors. The patient experienced clinical benefit following treatment with the combination of a BRAF and a MEK inhibitor. Rebiopsy of the tumor at a later time point, after BRAF and MEK inhibitors had been discontinued, showed loss of the AGAP3-BRAF fusion gene. Mixing experiments suggest that cells harboring both BRAFV600E and AGAP3-BRAF only have a fitness advantage over parental BRAFV600E cells during active treatment with a BRAF inhibitor.Conclusions: We report acquisition of a BRAF fusion as a novel mechanism of acquired resistance to vemurafenib in a patient with melanoma harboring a BRAFV600E mutation. The acquisition and regression of clones harboring this fusion during the presence and absence of a BRAF inhibitor are consistent with rapidly evolving clonal dynamics in melanoma. Clin Cancer Res; 23(18); 5631-8. ©2017 AACR.
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Malignant melanoma is among the most aggressive cancers and its incidence is increasing worldwide. Targeted therapies and immunotherapy have improved the survival of patients with metastatic melanoma in the last few years; however, available treatments are still unsatisfactory. While the role of the BRAF-MEK1/2-ERK1/2 pathway in melanoma is well established, the involvement of mitogen-activated protein kinases MEK5-ERK5 remains poorly explored. Here we investigated the function of ERK5 signaling in melanoma. We show that ERK5 is consistently expressed in human melanoma tissues and is active in melanoma cells. Genetic silencing and pharmacological inhibition of ERK5 pathway drastically reduce the growth of melanoma cells and xenografts harboring wild-type (wt) or mutated BRAF (V600E). We also found that oncogenic BRAF positively regulates expression, phosphorylation, and nuclear localization of ERK5. Importantly, ERK5 kinase and transcriptional transactivator activities are enhanced by BRAF. Nevertheless, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Accordingly, combination of MEK5 or ERK5 inhibitors with BRAFV600E inhibitor vemurafenib is more effective than single treatments in reducing colony formation and growth of BRAFV600E melanoma cells and xenografts. Overall, these data support a key role of the ERK5 pathway for melanoma growth in vitro and in vivo and suggest that targeting ERK5, alone or in combination with BRAF-MEK1/2 inhibitors, might represent a novel approach for melanoma treatment.
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The BRAF(V600E) mutation confers constitutive kinase activity and accounts for >90% of BRAF mutations in melanoma. This genetic alteration is a current therapeutic target; however, the antitumorigenic effects of the BRAF(V600E) inhibitor vemurafenib are short-lived and the majority of patients present tumor relapse in a short period after treatment. Characterization of vemurafenib resistance has been essential to the efficacy of next generation therapeutic strategies. Herein, we found that acute BRAF inhibition induced a decrease in active MMP-2, MT1-MMP and MMP-9, but did not modulate the metalloproteinase inhibitors TIMP-2 or RECK in naïve melanoma cells. In vemurafenib-resistant melanoma cells, we observed a lower growth rate and an increase in EGFR phosphorylation followed by the recovery of active MMP-2 expression, a mediator of cancer metastasis. Furthermore, we found a different profile of MMP inhibitor expression, characterized by TIMP-2 downregulation and RECK upregulation. In a 3D spheroid model, the invasion index of vemurafenib-resistant melanoma cells was more evident than in its non-resistant counterpart. We confirmed this pattern in a matrigel invasion assay and demonstrated that use of a matrix metalloproteinase inhibitor reduced the invasion of vemurafenib resistant melanoma cells but not drug naïve cells. Moreover, we did not observe a delimited group of cells invading the dermis in vemurafenib-resistant melanoma cells present in a reconstructed skin model. The same MMP-2 and RECK upregulation profile was found in this 3D skin model containing vemurafenib-resistant melanoma cells. Acute vemurafenib treatment induces the disorganization of collagen fibers and consequently, extracellular matrix remodeling, with this pattern observed even after the acquisition of resistance. Altogether, our data suggest that resistance to vemurafenib induces significant changes in the tumor microenvironment mainly by MMP-2 upregulation, with a corresponding increase in cell invasiveness.
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// Audrey Delmas 1, 2, 3 , Julia Cherier 1, 3 , Magdalena Pohorecka 1, 2, 3 , Claire Medale-Giamarchi 1, 2, 3 , Nicolas Meyer 1, 2, 4 , Anne Casanova 3 , Olivier Sordet 1, 3 , Laurence Lamant 1, 5 , Ariel Savina 6 , Anne Pradines 1, 2, 3 , Gilles Favre 1, 2, 3 1 Inserm, UMR 1037-CRCT, Toulouse, France 2 Université Paul Sabatier, Toulouse, France 3 Institut Claudius Regaud, Institut Universitaire du Cancer de Toulouse-Oncopole, Laboratory of Medical Biology and Oncogenetics, Toulouse, France 4 Centre Hospitalo-Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse-Oncopole, Department of Dermatology, Toulouse, France 5 Centre Hospitalo-Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse-Oncopole, Department of Pathology, Toulouse, France 6 Scientific Partnerships, Roche SAS, Boulogne Billancourt, France Correspondence to: Gilles Favre, e-mail: favre.gilles@iuct-oncopole.fr Keywords: melanoma, RHOB, AKT, vemurafenib, resistance Received: January 19, 2015 Accepted: April 25, 2015 Published: May 07, 2015 ABSTRACT The response of BRAF -mutant melanoma patients to BRAF inhibitors is dramatically impaired by secondary resistances and rapid relapse. So far, the molecular mechanisms driving these resistances are not completely understood. Here, we show that, in BRAF -mutant melanoma cells, inhibition of BRAF or its target MEK induces RHOB expression by a mechanism that depends on the transcription factor c-Jun. In those cells, RHOB deficiency causes hypersensitivity to BRAF and MEK inhibitors-induced apoptosis. Supporting these results, loss of RHOB expression in metastatic melanoma tissues is associated with an increased progression-free survival of BRAF -mutant patients treated with vemurafenib. Following BRAF inhibition, RHOB activates AKT whose inhibition causes hypersensitivity of BRAF -mutant melanoma cells to BRAF inhibitors. In mice, AKT inhibition synergizes with vemurafenib to block tumor growth of BRAF -mutant metastatic melanoma. Our findings reveal that BRAF inhibition activates a c-Jun/RHOB/AKT pathway that promotes tumor cell survival and further support a role of this pathway in the resistance of melanoma to vemurafenib. Our data also highlight the importance of using RHOB tumor levels as a biomarker to predict vemurafenib patient’s response and to select those that would benefit of the combination with AKT inhibitors.
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Abstract Melanoma is the most deadly form of skin cancer. An important feature of melanoma is its ability to metastasize and this contributes to its poor prognosis. RAS is mutated in ∼20% of melanomas and its downstream kinase BRAF is mutated in a further 50% of cases, these oncogenes regulate proliferation and survival through the MEK-ERK cascade. Selective inhibitors of BRAF such as vemurafenib have a remarkable clinical activity in patients with BRAF mutant melanomas with response rates of approximately 60-80%. In cells expressing activating BRAF mutations, vemurafenib blocks activation of the MAPK pathway. However, in cells expressing wild type BRAF and constitutively active upstream components that activate the MAPK pathway, BRAF inhibitors drive the paradoxical activation of this pathway. Here we show that kinase-dead BRAF promotes melanoma cell invasion and metastasis in the presence of oncogenic RAS. Notably, drugs that target BRAF mimic these effects and also induce RAS mutant melanoma cell invasion. The underlying mechanism is driven by paradoxical activation of the MEK-ERK pathway, resulting in increased secretion of the chemokine IL8 and subsequent secretion of extracellular matrix proteases, which induce invasion. We also show that BRAF inhibitors promote melanoma metastasis in an allograft model and that BRAF inhibitors induce invasion of drug-resistant BRAF mutant melanoma cells. We conclude that paradoxical activation of the MAPK pathway by BRAF inhibitors can drive melanoma invasion and metastasis in RAS mutant melanoma and in drug-resistant BRAF mutant melanoma cells. Citation Format: Berta Sanchez-Laorden, Amaya Viros, Maria Romina Girotti, Matthew Martin, Malin Pedersen, Alfonso Zambon, Dan Niculescu-Duvaz, Caroline Springer, Richard Marais. Paradoxical activation of the MEK/ERK pathway promotes melanoma invasion and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4307. doi:10.1158/1538-7445.AM2013-4307
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A large number of tumors shows a deregulation of the pathway RAS-RAF-MEK-ERK. Most of cases of melanoma are caused by the mutation V600E of BRAF, that leads to the constitutive activation of this kinase and of the MAPK pathway. One of the most important BRAF V600E inhibitor used against melanoma is vemurafenib.Extension study of melanoma patients with BRAF V600E tumors shows that vemurafenib treatment of these metastatic melanomas causes complete or partial tumor regression. However, the majority of patients eventually develops resistance or presents intrinsic resistance against this drug, and the tumor becomes more aggressive.Several mechanisms of resistance to BRAF inhibitors have been described. In most of these mechanisms the resistance to BRAF inhibitors results from reactivation of MEK-ERK pathway. Scaffold KSR2 is an important modulator of ERK-MAPK signalling pathway. In this study, we investigated the role of KSR2 in vemurafenib-treated melanoma cells.We found that treatment with the BRAF-selective inhibitor vemurafenib induced the expression of KSR2 in A375 human melanoma cells. Interestingly, the KSR2 overexpression increased the melanoma cells growth after treatment with vemurafenib. These results suggest that scaffold KSR2 could play an important role in the mechanism of resistance of melanoma against BRAF inhibitor vemurafenib.
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