RESUMO
Cytosolic caspase-8 is a mediator of death receptor signaling. While caspase-8 expression is lost in some tumors, it is increased in others, indicating a conditional pro-survival function of caspase-8 in cancer. Here, we show that tumor cells employ DNA-damage-induced nuclear caspase-8 to override the p53-dependent G2/M cell-cycle checkpoint. Caspase-8 is upregulated and localized to the nucleus in multiple human cancers, correlating with treatment resistance and poor clinical outcome. Depletion of caspase-8 causes G2/M arrest, stabilization of p53, and induction of p53-dependent intrinsic apoptosis in tumor cells. In the nucleus, caspase-8 cleaves and inactivates the ubiquitin-specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wild-type p53. This results in de facto p53 protein loss, switching cell fate from apoptosis toward mitosis. In summary, our work identifies a non-canonical role of caspase-8 exploited by cancer cells to override the p53-dependent G2/M cell-cycle checkpoint.
Assuntos
Caspase 8/metabolismo , Núcleo Celular/enzimologia , Proliferação de Células , Pontos de Checagem da Fase G2 do Ciclo Celular , Neoplasias/enzimologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/metabolismo , Antineoplásicos/farmacologia , Apoptose , Caspase 8/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Masculino , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Células PC-3 , Estabilidade Proteica , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/genéticaRESUMO
Elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been frequently reported in malignant melanoma suggesting that XIAP renders apoptosis resistance and thereby supports melanoma progression. Independent of its anti-apoptotic function, XIAP mediates cellular inflammatory signalling and promotes immunity against bacterial infection. The pro-inflammatory function of XIAP has not yet been considered in cancer. By providing detailed in vitro analyses, utilising two independent mouse melanoma models and including human melanoma samples, we show here that XIAP is an important mediator of melanoma neutrophil infiltration. Neutrophils represent a major driver of melanoma progression and are increasingly considered as a valuable therapeutic target in solid cancer. Our data reveal that XIAP ubiquitylates RIPK2, involve TAB1/RIPK2 complex and induce the transcriptional up-regulation and secretion of chemokines such as IL8, that are responsible for intra-tumour neutrophil accumulation. Alteration of the XIAP-RIPK2-TAB1 inflammatory axis or the depletion of neutrophils in mice reduced melanoma growth. Our data shed new light on how XIAP contributes to tumour growth and provides important insights for novel XIAP targeting strategies in cancer.
Assuntos
Proteínas Inibidoras de Apoptose , Melanoma , Infiltração de Neutrófilos , Neoplasias Cutâneas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Modelos Animais de Doenças , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/imunologia , Interleucina-8/biossíntese , Melanoma/genética , Melanoma/imunologia , Camundongos , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/imunologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/imunologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Genes, proteins, or cells influence each other and consequently create patterns, which can be increasingly better observed by experimental biology and medicine. Thereby, descriptive methods of statistics and bioinformatics sharpen and structure our perception. However, additionally considering the interconnectivity between biological elements promises a deeper and more coherent understanding of melanoma. For instance, integrative network-based tools and well-grounded inductive in silico research reveal disease mechanisms, stratify patients, and support treatment individualization. This review gives an overview of different modeling techniques beyond statistics, shows how different strategies align with the respective medical biology, and identifies possible areas of new computational melanoma research.
Assuntos
Simulação por Computador , Redes Reguladoras de Genes , Melanoma , Neoplasias Cutâneas , Biologia Computacional , Humanos , Melanoma/genética , Modelos Biológicos , Proteínas , Neoplasias Cutâneas/genéticaRESUMO
The transcription factor nuclear factor kappa-B (NFκB) is a key regulator of pro-inflammatory and pro-proliferative processes. Accordingly, uncontrolled NFκB activity may contribute to the development of severe diseases when the regulatory system is impaired. Since NFκB can be triggered by a huge variety of inflammatory, pro-and anti-apoptotic stimuli, its activation underlies a complex and tightly regulated signaling network that also includes multi-layered negative feedback mechanisms. Detailed understanding of this complex signaling network is mandatory to identify sensitive parameters that may serve as targets for therapeutic interventions. While many details about canonical and non-canonical NFκB activation have been investigated, less is known about cellular IκBα pools that may tune the cellular NFκB levels. IκBα has so far exclusively been described to exist in two different forms within the cell: stably bound to NFκB or, very transiently, as unbound protein. We created a detailed mathematical model to quantitatively capture and analyze the time-resolved network behavior. By iterative refinement with numerous biological experiments, we yielded a highly identifiable model with superior predictive power which led to the hypothesis of an NFκB-lacking IκBα complex that contains stabilizing IKK subunits. We provide evidence that other but canonical pathways exist that may affect the cellular IκBα status. This additional IκBα:IKKγ complex revealed may serve as storage for the inhibitor to antagonize undesired NFκB activation under physiological and pathophysiological conditions.
Assuntos
Bioquímica/métodos , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Biologia Computacional/métodos , Citosol/metabolismo , Humanos , Modelos Teóricos , Inibidor de NF-kappaB alfa , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Vanadatos/químicaRESUMO
Induction of DNA damage by UVB and UVA radiation may generate mutations and genomic instability leading to carcinogenesis. Therefore, skin cells being repeatedly exposed to ultraviolet (UV) light have acquired multilayered protective mechanisms to avoid malignant transformation. Besides extensive DNA repair mechanisms, the damaged skin cells can be eliminated by induction of apoptosis, which is mediated through the action of tumor suppressor p53. In order to prevent the excessive loss of skin cells and to maintain the skin barrier function, apoptotic pathways are counteracted by anti-apoptotic signaling including the AKT/mTOR pathway. However, AKT/mTOR not only prevents cell death, but is also active in cell cycle transition and hyper-proliferation, thereby also counteracting p53. In turn, AKT/mTOR is tuned down by the negative regulators being controlled by the p53. This inhibition of AKT/mTOR, in combination with transactivation of damage-regulated autophagy modulators, guides the p53-mediated elimination of damaged cellular components by autophagic clearance. Alternatively, p53 irreversibly blocks cell cycle progression to prevent AKT/mTOR-driven proliferation, thereby inducing premature senescence. Conclusively, AKT/mTOR via an extensive cross talk with p53 influences the UV response in the skin with no black and white scenario deciding over death or survival.
Assuntos
Carcinogênese/efeitos da radiação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pele/efeitos da radiação , Estresse Fisiológico/efeitos da radiação , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Carcinogênese/metabolismo , Senescência Celular/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Humanos , Camundongos , Transdução de Sinais/genética , Pele/imunologia , Pele/metabolismo , Neoplasias Cutâneas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Raios UltravioletaRESUMO
RL2 (recombinant lactaptin 2), a recombinant analogon of the human milk protein Κ-Casein, induces mitophagy and cell death in breast carcinoma cells. Furthermore, RL2 was shown to enhance extrinsic apoptosis upon long-term treatment while inhibiting it upon short-term stimulation. However, the effects of RL2 on the action of chemotherapeutic drugs that induce the intrinsic apoptotic pathway have not been investigated to date. Here, we examined the effects of RL2 on the doxorubicin (DXR)-induced cell death in breast cancer cells with three different backgrounds. In particular, we used BT549 and MDA-MB-231 triple-negative breast cancer (TNBC) cells, T47D estrogen receptor alpha (ERα) positive cells, and SKBR3 human epidermal growth factor receptor 2 (HER2) positive cells. BT549, MDA-MB-231, and T47D cells showed a severe loss of cell viability upon RL2 treatment, accompanied by the induction of mitophagy. Furthermore, BT549, MDA-MB-231, and T47D cells could be sensitized towards DXR treatment with RL2, as evidenced by loss of cell viability. In contrast, SKBR3 cells showed almost no RL2-induced loss of cell viability when treated with RL2 alone, and RL2 did not sensitize SKBR3 cells towards DXR-mediated loss of cell viability. Bioinformatic analysis of gene expression showed an enrichment of genes controlling metabolism in SKBR3 cells compared to the other cell lines. This suggests that the metabolic status of the cells is important for their sensitivity to RL2. Taken together, we have shown that RL2 can enhance the intrinsic apoptotic pathway in TNBC and ERα-positive breast cancer cells, paving the way for the development of novel therapeutic strategies.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Receptor alfa de Estrogênio , Linhagem Celular Tumoral , Apoptose , Doxorrubicina/farmacologiaRESUMO
Malignant melanoma (MM) is known to be intrinsically chemoresistant, even though only ~20% of MM carry mutations of the tumor suppressor p53. Despite improvement of systemic therapy the mortality rate of patients suffering from metastatic MM is still ~70%, highlighting the need for alternative treatment options or for the re-establishment of conventional therapeutic approaches, including chemotherapy. Screening the p53 mutation status in a cohort of 19 patient-derived melanoma samples, we identified one rarely described missense mutation of p53 leading to E285K amino acid exchange (mutp53(E285K)). Employing structural and computational analysis we revealed a major role of E285 residue in maintaining stable conformation of wild-type p53 (wtp53). E285K mutation was predicted to cause interruption of a salt-bridge network affecting the conformation of the C-terminal helix of the DNA-binding domain (DBD) thereby preventing DNA interaction. In this context, a cluster of frequently mutated amino acid residues in cancer was identified to putatively lead to similar structural effects as E285K substitution (E285 cluster). Functional analysis, including knockdown of endogenous p53 and reconstitution with diverse p53 missense mutants confirmed mutp53(E285K) to have lost transcriptional activity, to be localized in the cytosol of cancer cells, by both means conferring chemoresistance. Re-sensitization to cisplatin-induced cell death was achieved using clinically approved compounds aiming to restore p53 wild-type function (PRIMA1-Met), or inhibition of AKT-driven MAPK survival pathways (afuresertib), in both cases being partially due to ferroptosis induction. Consequently, active ferroptosis induction using the GPX4 inhibitor RSL3 proved superior in tumorselectively fighting MM cells. Due to high prevalence of the E285-cluster mutations in MM as well as in a variety of other tumor types, we conclude this cluster to serve an important function in tumor development and therapy and suggest new implications for ferroptosis induction in therapeutic applications fighting MM in particular and cancer in general.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma , Neoplasias Cutâneas , Proteína Supressora de Tumor p53 , Humanos , Aminoácidos , Linhagem Celular Tumoral , Citosol/metabolismo , DNA , Resistencia a Medicamentos Antineoplásicos/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Mutação , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Despite high initial response rates to targeted kinase inhibitors, the majority of patients suffering from metastatic melanoma present with high relapse rates, demanding for alternative therapeutic options. We have previously developed a drug repurposing workflow to identify metabolic drug targets that, if depleted, inhibit the growth of cancer cells without harming healthy tissues. In the current study, we have applied a refined version of the workflow to specifically predict both, common essential genes across various cancer types, and melanoma-specific essential genes that could potentially be used as drug targets for melanoma treatment. The in silico single gene deletion step was adapted to simulate the knock-out of all targets of a drug on an objective function such as growth or energy balance. Based on publicly available, and in-house, large-scale transcriptomic data metabolic models for melanoma were reconstructed enabling the prediction of 28 candidate drugs and estimating their respective efficacy. Twelve highly efficacious drugs with low half-maximal inhibitory concentration values for the treatment of other cancers, which are not yet approved for melanoma treatment, were used for in vitro validation using melanoma cell lines. Combination of the top 4 out of 6 promising candidate drugs with BRAF or MEK inhibitors, partially showed synergistic growth inhibition compared to individual BRAF/MEK inhibition. Hence, the repurposing of drugs may enable an increase in therapeutic options e.g., for non-responders or upon acquired resistance to conventional melanoma treatments.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Humanos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Recidiva Local de Neoplasia/tratamento farmacológico , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinases de Proteína Quinase Ativadas por Mitógeno , Desenvolvimento de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular TumoralRESUMO
Dabrafenib inhibits the cell proliferation of metastatic melanoma with the oncogenic BRAF(V600)-mutation. However, dabrafenib monotherapy is associated with pERK reactivation, drug resistance, and consequential relapse. A clinical drug-dose determination study shows increased pERK levels upon daily administration of more than 300 mg dabrafenib. To clarify whether such elevated drug concentrations could be reached by long-term drug accumulation, we mechanistically coupled the pharmacokinetics (MCPK) of dabrafenib and its metabolites. The MCPK model is qualitatively based on in vitro and quantitatively on clinical data to describe occupancy-dependent CYP3A4 enzyme induction, accumulation, and drug-drug interaction mechanisms. The prediction suggests an eight-fold increase in the steady-state concentration of potent desmethyl-dabrafenib and its inactive precursor carboxy-dabrafenib within four weeks upon 150 mg b.d. dabrafenib. While it is generally assumed that a higher dose is not critical, we found experimentally that a high physiological dabrafenib concentration fails to induce cell death in embedded 451LU melanoma spheroids.
RESUMO
Despite remarkable advances in therapeutic interventions, malignant melanoma (MM) remains a life-threating disease. Following high initial response rates to targeted kinase-inhibition metastases quickly acquire resistance and present with enhanced tumor progression and invasion, demanding alternative treatment options. We show 2nd generation hexameric TRAIL-receptor-agonist IZI1551 (IZI) to effectively induce apoptosis in MM cells irrespective of the intrinsic BRAF/NRAS mutation status. Conditioning to the EC50 dose of IZI converted the phenotype of IZI-sensitive parental MM cells into a fast proliferating and invasive, IZI-resistant metastasis. Mechanistically, we identified focal adhesion kinase (FAK) to play a dual role in phenotype-switching. In the cytosol, activated FAK triggers survival pathways in a PI3K- and MAPK-dependent manner. In the nucleus, the FERM domain of FAK prevents activation of wtp53, as being expressed in the majority of MM, and consequently intrinsic apoptosis. Caspase-8-mediated cleavage of FAK as well as FAK knockdown, and pharmacological inhibition, respectively, reverted the metastatic phenotype-switch and restored IZI responsiveness. FAK inhibition also re-sensitized MM cells isolated from patient metastasis that had relapsed from targeted kinase inhibition to cell death, irrespective of the intrinsic BRAF/NRAS mutation status. Hence, FAK-inhibition alone or in combination with 2nd generation TRAIL-receptor agonists may be recommended for treatment of initially resistant and relapsed MM, respectively.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Linhagem Celular Tumoral , Quinase 1 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais/fisiologia , Neoplasias Cutâneas , Melanoma Maligno CutâneoRESUMO
Primary or acquired therapy resistance is a major obstacle to the effective treatment of cancer. Resistance to apoptosis has long been thought to contribute to therapy resistance. We show here that recombinant TRAIL and CDK9 inhibition cooperate in killing cells derived from a broad range of cancers, importantly without inducing detectable adverse events. Remarkably, the combination of TRAIL with CDK9 inhibition was also highly effective on cancers resistant to both, standard-of-care chemotherapy and various targeted therapeutic approaches. Dynamic BH3 profiling revealed that, mechanistically, combining TRAIL with CDK9 inhibition induced a drastic increase in the mitochondrial priming of cancer cells. Intriguingly, this increase occurred irrespective of whether the cancer cells were sensitive or resistant to chemo- or targeted therapy. We conclude that this pro-apoptotic combination therapy has the potential to serve as a highly effective new treatment option for a variety of different cancers. Notably, this includes cancers that are resistant to currently available treatment modalities.
Assuntos
Antineoplásicos , Neoplasias , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Mitocôndrias , Neoplasias/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Induction of apoptosis of keratinocytes by ultraviolet (UV) radiation is a protective phenomenon relevant in limiting the survival of cells with irreparable DNA damage. Changes in UV-induced apoptosis may therefore have significant impact on photocarcinogenesis. We have found that the immunomodulatory cytokine IL-12 suppresses UV-mediated apoptosis of keratinocytes both in vitro and in vivo. IL-12 caused a remarkable reduction in UV-specific DNA lesions which was due to induction of DNA repair. In accordance with this, IL-12 induced the expression of particular components of the nucleotide-excision repair complex. Our results show that cytokines can protect cells from apoptosis induced by DNA-damaging UV radiation by inducing DNA repair, and that nucleotide-excision repair can be manipulated by cytokines.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Interleucina-12/farmacologia , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Adjuvantes Imunológicos/farmacologia , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Humanos , Neoplasias Induzidas por Radiação/patologia , Neoplasias Induzidas por Radiação/prevenção & controle , Proteínas Recombinantes/farmacologia , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/prevenção & controle , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
Glucocorticoids (GC), synthesized by the 11ß-hydroxylase (Cyp11b1), control excessive inflammation through immunosuppressive actions. The skin was proposed to regulate homeostasis by autonomous GC production in keratinocytes. However, their immunosuppressive capacity and clinical relevance remain unexplored. Here, we demonstrate the potential of skin-derived GC and their role in the regulation of physiological and prevalent inflammatory skin conditions. In line with 11ß-hydroxylase deficiency in human inflammatory skin disorders, genetic in vivo Cyp11b1 ablation and long-term GC deficiency in keratinocytes primed the murine skin immune system resulting in spontaneous skin inflammation. Deficient skin GC in experimental models for inflammatory skin disorders led to exacerbated contact hypersensitivity and psoriasiform skin inflammation accompanied by decreased regulatory T cells and the involvement of unconventional T cells. Our findings provide insights on how skin homeostasis and pathology are critically regulated by keratinocyte-derived GC, emphasizing the immunoregulatory potential of endogenous GC in the regulation of epithelial immune microenvironment.
Assuntos
Glucocorticoides , Esteroide 11-beta-Hidroxilase , Animais , Homeostase , Humanos , Inflamação/patologia , Queratinócitos , Camundongos , Pele/patologiaRESUMO
Second generation TRAIL-based therapeutics, combined with sensitising co-treatments, have recently entered clinical trials. However, reliable response predictors for optimal patient selection are not yet available. Here, we demonstrate that a novel and translationally relevant hexavalent TRAIL receptor agonist, IZI1551, in combination with Birinapant, a clinically tested IAP antagonist, efficiently induces cell death in various melanoma models, and that responsiveness can be predicted by combining pathway analysis, data-driven modelling and pattern recognition. Across a panel of 16 melanoma cell lines, responsiveness to IZI1551/Birinapant was heterogeneous, with complete resistance and pronounced synergies observed. Expression patterns of TRAIL pathway regulators allowed us to develop a combinatorial marker that predicts potent cell killing with high accuracy. IZI1551/Birinapant responsiveness could be predicted not only for cell lines, but also for 3D tumour cell spheroids and for cells directly isolated from patient melanoma metastases (80-100% prediction accuracies). Mathematical parameter reduction identified 11 proteins crucial to ensure prediction accuracy, with x-linked inhibitor of apoptosis protein (XIAP) and procaspase-3 scoring highest, and Bcl-2 family members strongly represented. Applied to expression data of a cohort of n = 365 metastatic melanoma patients in a proof of concept in silico trial, the predictor suggested that IZI1551/Birinapant responsiveness could be expected for up to 30% of patient tumours. Overall, response frequencies in melanoma models were very encouraging, and the capability to predict melanoma sensitivity to combinations of latest generation TRAIL-based therapeutics and IAP antagonists can address the need for patient selection strategies in clinical trials based on these novel drugs.
Assuntos
Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Reconhecimento Automatizado de Padrão , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dipeptídeos/farmacologia , Humanos , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Metástase NeoplásicaRESUMO
BACKGROUND: Melanoma is the most aggressive and deadly form of skin cancer with increasing case numbers worldwide. The development of inhibitors targeting mutated BRAF (found in around 60% of melanoma patients) has markedly improved overall survival of patients with late-stage tumors, even more so when combined with MEK inhibitors targeting the same signaling pathway. However, invariably patients become resistant to this targeted therapy resulting in rapid progression with treatment-refractory disease. The purpose of this study was the identification of new kinase inhibitors that do not lead to the development of resistance in combination with BRAF inhibitors (BRAFi), or that could be of clinical benefit as a 2nd line treatment for late-stage melanoma patients that have already developed resistance. METHODS: We have screened a 274-compound kinase inhibitor library in 3 BRAF mutant melanoma cell lines (each one sensitive or made resistant to 2 distinct BRAFi). The screening results were validated by dose-response studies and confirmed the killing efficacies of many kinase inhibitors. Two different tools were applied to investigate and quantify potential synergistic effects of drug combinations: the Chou-Talalay method and the Synergyfinder application. In order to exclude that resistance to the new treatments might occur at later time points, synergistic combinations were administered to fluorescently labelled parental and resistant cells over a period of > 10 weeks. RESULTS: Eight inhibitors targeting Wee1, Checkpoint kinase 1/2, Aurora kinase, MEK, Polo-like kinase, PI3K and Focal adhesion kinase killed melanoma cells synergistically when combined with a BRAFi. Additionally, combination of a Wee1 and Chk inhibitor showed synergistic killing effects not only on sensitive cell lines, but also on intrinsically BRAFi- and treatment induced-resistant melanoma cells. First in vivo studies confirmed these observations. Interestingly, continuous treatment with several of these drugs, alone or in combination, did not lead to emergence of resistance. CONCLUSIONS: Here, we have identified new, previously unexplored (in the framework of BRAFi resistance) inhibitors that have an effect not only on sensitive but also on BRAFi-resistant cells. These promising combinations together with the new immunotherapies could be an important step towards improved 1st and 2nd line treatments for late-stage melanoma patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Melanoma/tratamento farmacológico , Inibidores de Proteínas Quinases/isolamento & purificação , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Melanoma/fisiopatologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
BACKGROUND: Metabolic rewiring allows cancer cells to sustain high proliferation rates. Thus, targeting only the cancer-specific cellular metabolism will safeguard healthy tissues. METHODS: We developed the very efficient FASTCORMICS RNA-seq workflow (rFASTCORMICS) to build 10,005 high-resolution metabolic models from the TCGA dataset to capture metabolic rewiring strategies in cancer cells. Colorectal cancer (CRC) was used as a test case for a repurposing workflow based on rFASTCORMICS. FINDINGS: Alternative pathways that are not required for proliferation or survival tend to be shut down and, therefore, tumours display cancer-specific essential genes that are significantly enriched for known drug targets. We identified naftifine, ketoconazole, and mimosine as new potential CRC drugs, which were experimentally validated. INTERPRETATION: The here presented rFASTCORMICS workflow successfully reconstructs a metabolic model based on RNA-seq data and successfully predicted drug targets and drugs not yet indicted for colorectal cancer. FUND: This study was supported by the University of Luxembourg (IRP grant scheme; R-AGR-0755-12), the Luxembourg National Research Fund (FNR PRIDE PRIDE15/10675146/CANBIO), the Fondation Cancer (Luxembourg), the European Union's Horizon2020 research and innovation programme under the Marie Sklodowska- Curie grant agreement No 642295 (MEL-PLEX), and the German Federal Ministry of Education and Research (BMBF) within the project MelanomSensitivity (BMBF/BM/7643621).
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais , Biologia Computacional , Descoberta de Drogas , Metabolismo Energético/efeitos dos fármacos , Terapia de Alvo Molecular , Algoritmos , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Deleção de Genes , Perfilação da Expressão Gênica , Humanos , Reprodutibilidade dos Testes , Fluxo de TrabalhoRESUMO
Malignant melanoma is a highly aggressive form of skin cancer responsible for the majority of skin cancer-related deaths. Recent insight into the heterogeneous nature of melanoma suggests more personalised treatments may be necessary to overcome drug resistance and improve patient care. To this end, reliable molecular signatures that can accurately predict treatment responsiveness need to be identified. In this study, we applied multiplex phosphoproteomic profiling across a panel of 24 melanoma cell lines with different disease-relevant mutations, to predict responsiveness to MEK inhibitor trametinib. Supported by multivariate statistical analysis and multidimensional pattern recognition algorithms, the responsiveness of individual cell lines to trametinib could be predicted with high accuracy (83% correct predictions), independent of mutation status. We also successfully employed this approach to case specifically predict whether individual melanoma cell lines could be sensitised to trametinib. Our predictions identified that combining MEK inhibition with selective targeting of c-JUN and/or FAK, using siRNA-based depletion or pharmacological inhibitors, sensitised resistant cell lines and significantly enhanced treatment efficacy. Our study indicates that multiplex proteomic analyses coupled with pattern recognition approaches could assist in personalising trametinib-based treatment decisions in the future.
Assuntos
Antineoplásicos/farmacologia , Melanoma/tratamento farmacológico , Piridonas/farmacologia , Pirimidinonas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Melanoma/metabolismo , Melanoma/patologiaRESUMO
Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.
Assuntos
Transformação Celular Neoplásica/genética , Melanoma/diagnóstico , Neoplasias Cutâneas/diagnóstico , Esferoides Celulares/patologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Humanos , Melanoma/patologia , Neoplasias Cutâneas/patologia , Melanoma Maligno CutâneoRESUMO
Single-chain formats of TNF-related apoptosis inducing ligand (scTRAIL) can serve as effector components of tumour-associated antigen-targeted as well as non-targeted fusion proteins, being characterized by high tumour cell-specific induction of apoptosis through death receptor activation. We studied the suitability of immunoglobulin G as a scaffold for oligovalent and bispecific TRAIL fusion proteins. Thus, we developed novel targeted hexa- and dodecavalent IgG-scTRAIL molecules by fusing scTRAIL to the C-terminus of either light (LC-scTRAIL) or heavy immunoglobulin chain (HC-scTRAIL), or to both ends (LC/HC-scTRAIL) of the anti-EGFR IgG antibody hu225. The binding specificity to EGFR and death receptors was retained in all IgG-scTRAIL formats and translated into high antigen-specific bioactivity on EGFR-positive Colo205, HCT116 and WM1366 tumour cell lines, with or without sensitization to apoptosis by bortezomib. In vivo, therapeutic potential was assessed for one of the targeted variants, HC-scTRAIL, compared to the non-targeted Fc-scTRAIL. Both molecules showed a significant reduction of tumour volume and synergism with a Smac mimetic in a Colo205 xenograft tumour model. The IgG-scTRAIL format allows directing a defined, highly bioactive form of TRAIL to a wide variety of tumour antigens, enabling customized solutions for a patient-specific targeted cancer therapy with a reduced risk of side effects.
Assuntos
Imunoglobulina G/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Receptores ErbB/imunologia , Humanos , Imunoglobulina G/química , Ligante Indutor de Apoptose Relacionado a TNF/químicaRESUMO
Metastatic melanoma remains a life-threatening disease because most tumors develop resistance to targeted kinase inhibitors thereby regaining tumorigenic capacity. We show the 2nd generation hexavalent TRAIL receptor-targeted agonist IZI1551 to induce pronounced apoptotic cell death in mutBRAF melanoma cells. Aiming to identify molecular changes that may confer IZI1551 resistance we combined Dynamic Bayesian Network modelling with a sophisticated regularization strategy resulting in sparse and context-sensitive networks and show the performance of this strategy in the detection of cell line-specific deregulations of a signalling network. Comparing IZI1551-sensitive to IZI1551-resistant melanoma cells the model accurately and correctly predicted activation of NFκB in concert with upregulation of the anti-apoptotic protein XIAP as the key mediator of IZI1551 resistance. Thus, the incorporation of multiple regularization functions in logical network optimization may provide a promising avenue to assess the effects of drug combinations and to identify responders to selected combination therapies.