RESUMO
Truncated O-GalNAc glycosylation is an important feature of pancreatic ductal adenocarcinomas (PDAC) and expression of truncated O-GalNAc glycans is strongly associated with decreased survival and poor prognosis. It has been proven, that aberrant O-GalNAc glycosylation influence PDAC signaling to promote oncogenic properties, but elucidation of the influence of truncated O-GalNAc glycosylation on different signaling molecules has just been started. We herein elucidated the impact of aberrant O-GalNAc glycosylation on two important PDAC signaling pathways, namely AKT/mTOR and RAS/MAPK. In PDAC cells expressing truncated O-GalNAc glycans, we identified differentially expressed proteins associated with AKT/mTOR and RAS/MAPK pathways using quantitative proteomics. Since AKT, a key-signaling molecule in PDAC, was among the identified proteins, we analyzed AKT and found a strikingly enhanced S473 phosphorylation and identified a previously unknown O-GalNAc-modification. Consecutive analysis of COSMC knockdowns in PDAC revealed strong effects on AKT upstream and downstream effector molecules. Interestingly, truncated O-GalNAc glycans could facilitate an mTORC1 inhibitor resistance using AZD8055. In addition, as AKT/mTOR pathway has extensive cross talks with RAS/MAPK pathway we analyzed the pathways and found it negatively regulated. Finally, we found that the expression of epithelial-mesenchymal-transition markers, key features of aggressive PDACs cells, are enhanced and truncated O-GalNAc glycans enhance pancreatic cancer cell growth in a xenograft mouse model. Our study demonstrates that truncated O-GalNAc glycans have a strong impact on AKT/mTOR and RAS/MAPK signaling pathways, are modulated by EGF or IGF-1 signaling and should be considered for targeted therapy of these pathways in PDAC.
RESUMO
MALT1 is a key mediator of NF-κB signaling and a main driver of B-cell lymphomas. Remarkably, MALT1 is expressed in the majority of pancreatic ductal adenocarcinomas (PDACs) as well, but absent from normal exocrine pancreatic tissue. Following, MALT1 shows off to be a specific target in cancer cells of PDAC without affecting regular pancreatic cells. Therefore, we studied the impact of pharmacological MALT1 inhibition in pancreatic cancer and showed promising effects on tumor progression. Mepazine (Mep), a phenothiazine derivative, is a known potent MALT1 inhibitor. Newly, we described that biperiden (Bip) is a potent MALT1 inhibitor with even less pharmacological side effects. Thus, Bip is a promising drug leading to reduced proliferation and increased apoptosis in PDAC cells in vitro and in vivo. By compromising MALT1 activity, nuclear translocation of c-Rel is prevented. c-Rel is critical for NF-κB-dependent inhibition of apoptosis. Hence, off-label use of Bip or Mep represents a promising new therapeutic approach to PDAC treatment. Regularly, the Anticholinergicum Bip is used to treat neurological side effects of Phenothiazines, like extrapyramidal symptoms.
Assuntos
Biperideno/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Fenotiazinas/farmacologia , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/biossíntese , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/química , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , NF-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-rel/metabolismo , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Epithelial-to-mesenchymal transition (EMT) and cancer stem cells (CSC) contribute to tumour progression and metastasis. Assessment of transcription factors involved in these two mechanisms can help to identify new targets for an oncological therapy. In this study, we focused on the evaluation of the transcription factor Six1 (Sine oculis 1). This protein is involved in embryologic development and its contribution to carcinogenesis has been described in several studies. METHODS: Immunohistochemistry against Six1 was performed on a tissue microarray containing specimens of primary pancreatic ductal adenocarcinomas (PDAC) of 139 patients. Nuclear and cytoplasmic expression was evaluated and correlated to histopathological parameters. Expression of Six1 was inhibited transiently by siRNA in Panc1 and BxPc3 cells and stably by shRNA in Panc1 cells. Expression analysis of CDH1 and Vimentin mRNA was performed and cell motility was tested in a migration assay. Panc1 cells transfected with Six1 shRNA or scrambled shRNA were injected subcutaneously into nude mice. Tumour growth was observed for four weeks. Afterwards, tumours were stained against Six1, CD24 and CD44. RESULTS: Six1 was overexpressed in the cytoplasm and cellular nuclei in malignant tissues (p < 0.0001). No correlation to histopathological parameters could be detected. Six1 down-regulation decreased pancreatic cancer cell motility in vitro. CDH1 and vimentin expression was decreased after inhibition of the expression of Six1. Pancreatic tumours with impaired expression of Six1 showed significantly delayed growth and displayed loss of the CD24+/CD44+ phenotype. CONCLUSION: We show that Six1 is overexpressed in human PDAC and that its inhibition results in a decreased tumour progression in vitro and in vivo. Therefore, targeting Six1 might be a novel therapeutic approach in patients with pancreatic cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Interferência de RNA , Animais , Transição Epitelial-Mesenquimal , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias PancreáticasRESUMO
BACKGROUND: Human pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies in the world and despite great efforts in research types of treatment remain limited. A frequently detected alteration in PDACs is a truncated O-linked N-acetylgalactosamine (GalNAc) glycosylation with expression of the Tn antigen. Changes in O-glycosylation affect posttranslationally modified O-GalNAc proteins resulting in profound cellular alterations. Tn antigen is a tumor associated glycan detected in 75-90 % of PDACs and up to 67 % in its precursor lesions. Since the role of Tn antigen expression in PDAC is insufficiently understood we analyzed the impact of COSMC mediated Tn antigen expression in two human PDAC cell lines on cellular oncogenic properties. METHODS: Forced expression of Tn antigen on O-glycosylated proteins in pancreatic cancer cells was induced by lentiviral-mediated knockdown of the COSMC chaperone, which prevented O-glycan elongation beyond the initial GalNAcα1- residue on O-linked glycoproteins. Altered O-GalNAc glycosylation was analyzed in human pancreatic cancer cell lines Panc-1 and L3.6pl using Western and Far-Western blot as well as immunocytochemical techniques. To assess the biological implications of COSMC function on oncogenic properties, cell viability assays, scratch assays combined with live cell imaging, migration and apoptosis assays were performed. Lectin based glycoprotein enrichment with subsequent mass spectrometric analysis identified new cancer O-GalNAc modified proteins. Expression of Tn antigen bearing Nucleolin in patient derived PDAC tumor specimens was evaluated and correlated with clinicopathological data. RESULTS: Tn antigen expression was induced on various O-GalNAc glycoproteins in COSMC deficient cell lines compared to the control. Proliferation was reduced (p < 0.001) in COSMC knockdown cells, whereas migration was increased (p < 0.001) and apoptosis was decreased (p = 0.03), highlighting the importance of Tn antigen expression on metastatic and anti-apoptotic behavior of PDAC derived cells. Nucleolin was identified as O-GalNAc modified protein in COSMC deficient PDAC cell lines. Interestingly, immunohistochemical staining and co-localization studies of patient derived PDACs revealed poor survival for patients with strong co-localization of Tn antigen and Nucleolin (p = 0.037). CONCLUSION: This study substantiates the influence of altered O-glycan (Tn/STn) expression on oncogenic properties in pancreatic cancer and identifies O-GalNAc modified Nucleolin as novel prognostic marker.
Assuntos
Carcinogênese/patologia , Técnicas de Silenciamento de Genes , Chaperonas Moleculares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Antígenos Glicosídicos Associados a Tumores , Carcinogênese/genética , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Glicosilação , Humanos , Espectrometria de Massas , Chaperonas Moleculares/metabolismo , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/enzimologia , Fosfoproteínas/metabolismo , Polissacarídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , NucleolinaRESUMO
UNLABELLED: Until today, there is no systemic treatment available for advanced cholangiocarcinoma (CCA). Recent studies have shown a frequent upregulation of the PI3K-AKT-mTOR and RAF-MEK-ERK pathways in this type of cancer. However, considering their high extend of redundancy and cross-talk, targeting only one pathway is likely to result in therapy failure and emergence of resistances. To provide a rationale for treatment of CCA with inhibitors of these respective pathways, we analyzed the effects of AKT inhibitor MK-2206, MEK inhibitor AZD6244 (ARRY-142886) and mTOR kinase inhibitor AZD8055 on three CCA cell lines in vitro, concerning proliferation, cell signaling and apoptosis. Furthermore, AZD6244 resistant cell lines have been generated to investigate, how their response may be affected by prolonged treatment with only a single inhibitor. Our data demonstrates that co-targeting of both, the PI3K/AKT/mTOR and RAF-MEK-ERK pathway, as well as vertical targeting of AKT and mTOR results in strong synergistic effects on proliferation and cell survival with combination indices below 0.3. Mechanistically, the combinatorial treatment with MK-2206 in addition to AZD8055 is necessary because AKT kinase activity was quickly restored after mTOR kinase inhibition. Interestingly, acquired MEK inhibitor resistance to AZD6244 was reversed by combined treatment with AZD6244 and either MK-2206 or AZD8055. Our data suggest that a combination of inhibitors targeting those respective pathways may be a viable approach for future application in patients with cholangiocarcinoma. IMPLICATIONS: AKT, mTOR and MEK are promising targets for a combinatorial treatment of cholangiocarcinoma cells even after acquisition of MEK inhibitor resistance.
Assuntos
Antineoplásicos/farmacologia , Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Benzimidazóis/farmacologia , Ductos Biliares Intra-Hepáticos/metabolismo , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Quinases raf/antagonistas & inibidoresRESUMO
Cholangiocarcinoma (CCA) is a rare, but devastating disease arising from the epithelium of intrahepatic and extrahepatic bile ducts. There are neither effective systemic therapies nor satisfying treatment options for inoperable CCA. Histopathological and biochemical studies of CCA show frequent dysregulation of the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) pathway. Therefore, we investigated the efficacy of the mTOR inhibitor RAD001 and the impact of AKT signaling following mTOR inhibition in the treatment of CCA. RAD001 significantly inhibits proliferation of CCA cell lines, however, a concentration-dependent and isoform specific feedback activation of the three AKT isoforms (AKT1, AKT2 and AKT3) was observed after mTOR inhibition. As activation of AKT might limit the RAD001-mediated anti-tumor effect, the efficacy of combined mTOR and AKT inhibition was investigated using the allosteric AKT inhibitor MK-2206. Our results show that inhibition of AKT potentiates the efficacy of mTOR inhibition both in vitro and in a xenograft mouse model in vivo. Mechanistically, the antiproliferative effect of the pan-AKT inhibitor MK2206 in the CCA cell line TFK-1 was due to inhibition of AKT1 and AKT2, because knockdown of either AKT1 or AKT2, but not AKT3, showed a synergistic reduction of cell proliferation in combination with mTOR treatment. Finally, using an AKT isoform specific in vitro kinase assay, enzymatic activity of each of the three AKT isoforms was detected in all tissue samples from CCA patients, analyzed. In summary, our preclinical data suggest that combined targeting of mTOR and AKT using RAD001 and MK-2206 might be a new, effective strategy for the treatment of CCA.
Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Ductos Biliares Intra-Hepáticos/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Sinergismo Farmacológico , Everolimo , Citometria de Fluxo , Humanos , Imunoprecipitação , Camundongos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
Cancer-specific mutations in the iSH2 (inter-SH2) and nSH2 (N-terminal SH2) domains of p85alpha, the regulatory subunit of phosphatidylinositide 3-kinase (PI3K), show gain of function. They induce oncogenic cellular transformation, stimulate cellular proliferation, and enhance PI3K signaling. Quantitative determinations of oncogenic activity reveal large differences between individual mutants of p85alpha. The mutant proteins are still able to bind to the catalytic subunits p110alpha and p110beta. Studies with isoform-specific inhibitors of p110 suggest that expression of p85 mutants in fibroblasts leads exclusively to an activation of p110alpha, and p110alpha is the sole mediator of p85 mutant-induced oncogenic transformation. The characteristics of the p85 mutants are in agreement with the hypothesis that the mutations weaken an inhibitory interaction between p85alpha and p110alpha while preserving the stabilizing interaction between p85alpha iSH2 and the adapter-binding domain of p110alpha.
Assuntos
Transformação Celular Neoplásica/genética , Mutação , Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Adenina/análogos & derivados , Adenina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Domínio Catalítico/genética , Linhagem Celular , Proliferação de Células , Células Cultivadas , Embrião de Galinha , Classe I de Fosfatidilinositol 3-Quinases , Dioxóis/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Imunoprecipitação , Morfolinas/farmacologia , Neoplasias/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ligação Proteica , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pirimidinonas/farmacologia , Quinazolinas/farmacologia , Tiazolidinedionas/farmacologia , TransfecçãoRESUMO
BACKGROUND: Due to the frequent dysregulation of the PI3K/AKT/mTOR signaling pathway, mTOR represents a suitable therapeutic target in hepatocellular carcinoma (HCC). However, emerging data from clinical trials of HCC patients indicate that mTOR inhibition by RAD001 (Everolimus) alone has only moderate antitumor efficacy which may be due to the feedback activation of AKT after mTOR inhibition. In this study, we analyzed the effects of dual inhibition of mTOR and AKT on the proliferation of HCC cell lines. In addition, we measured the feedback activation of each of the AKT isoforms after mTOR inhibition in HCC cell lines and their enzymatic activity in primary samples from HCC patients. METHODS: The activation status of specific AKT isoforms in human HCC samples and corresponding healthy liver tissue was analyzed using an AKT isoform specific in vitro kinase assay. AKT isoform activation after mTOR inhibition was analyzed in three HCC cell lines (Hep3B, HepG2 and Huh7), and the impact of AKT signaling on proliferation after mTOR inhibition was investigated using the novel AKT inhibitor MK-2206 and AKT isoform specific knockdown cells. RESULTS: AKT isoforms become differentially activated during feedback activation following RAD001 treatment. The combination of mTOR inhibition and AKT isoform knockdown showed only a weak synergistic effect on proliferation of HCC cell lines. However, the combinatorial treatment with RAD001 and the pan AKT inhibitor MK-2206 resulted in a strong synergism, both in vitro and in vivo. Moreover, by analyzing primary HCC tissue samples we were able to demonstrate that a hotspot mutation (H1047R) of PI3KCA, the gene encoding the catalytic subunit of PI3K, was associated with increased in vitro kinase activity of all AKT isoforms in comparison to healthy liver tissue of the patient. CONCLUSION: Our results demonstrate that dual targeting of mTOR and AKT by use of RAD001 and the pan AKT inhibitor MK-2206 does effectively inhibit proliferation of HCC cell lines. These data suggest that combined treatment with RAD001 and MK-2206 may be a promising therapy approach in the treatment of hepatocellular carcinoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Sinergismo Farmacológico , Everolimo , Retroalimentação Fisiológica , Feminino , Técnicas de Silenciamento de Genes , Células Hep G2 , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias Hepáticas/química , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos SCID , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sirolimo/administração & dosagem , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Cosmc is ubiquitously expressed and acts as a specific molecular chaperone assisting the folding and stability of core 1 synthase. Thus, it plays a crucial role in the biosynthesis of O-linked glycosylation of proteins. Here, we show that ablation of Cosmc in the exocrine pancreas of mice causes expression of truncated O-glycans (Tn antigen), resulting in exocrine pancreatic insufficiency with decreased activities of digestive enzymes and diabetes. To understand the molecular causes of the pleiotropic phenotype, we used Vicia villosa agglutinin to enrich Tn antigen-modified proteins from Cosmc-KO pancreatic lysates and performed a proteomic analysis. Interestingly, a variety of proteins were identified, of which bile salt-activated lipase (also denoted carboxyl-ester lipase, Cel) was the most abundant. In humans, frameshift mutations in CEL cause maturity-onset diabetes of the young type 8 (MODY8), a monogenic syndrome of diabetes and pancreatic exocrine dysfunction. Here, we provide data suggesting that differentially O-glycosylated Cel could negatively affect beta cell function. Taken together, our findings demonstrate the importance of correct O-glycan formation for normal exocrine and endocrine pancreatic function, implying that aberrant O-glycans might be relevant for pathogenic mechanisms of the pancreas.
Assuntos
Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Pâncreas Exócrino/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Glicosilação , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Proteoma , Proteômica/métodosRESUMO
BACKGROUND: Development of small molecular inhibitors against BRAF and MEK has been a breakthrough in the treatment of malignant melanoma. However, the long-term effect is foiled in virtually all patients by the emergence of resistant tumor cell populations. Therefore, mechanisms resulting in the acquired resistance against BRAF and MEK inhibitors have gained much attention and several strategies have been proposed to overcome tumor resistance, including interval treatment or withdrawal of these compounds after disease progression. METHODS: Using a panel of cell lines with an acquired resistance against MEK inhibitors, we have evaluated the sensitivity of these cells against compounds targeting AKT/mTOR signaling, as well as novel ERK1/2 inhibitors. Furthermore, the effects of withdrawal of MEK inhibitor on migration in resistant cell lines were analyzed. RESULTS: We demonstrate that withdrawal of BRAF or MEK inhibitors in tumor cells with an acquired resistance results in reactivation of ERK1/2 signaling and upregulation of EMT-inducing transcription factors, leading to a highly migratory and invasive phenotype of cancer cells. Furthermore, we show that migration in these cells is independent from AKT/mTOR signaling. However, combined targeting of AKT/mTOR using MK-2206 and AZD8055 efficiently inhibits proliferation in all resistant tumor cell lines analyzed. CONCLUSIONS: We propose that combined targeting of MEK/AKT/mTOR or treatment with a novel ERK1/2 inhibitor downstream of BRAF/MEK suppresses proliferation as well as migration and invasion in resistant tumor cells. We provide a rationale against the discontinuation of BRAF or MEK inhibitors in patients with an acquired resistance, and provide a rationale for combined targeting of AKT/mTOR and MEK/ERK1/2, or direct targeting of ERK1/2 as an effective treatment strategy.
Assuntos
Movimento Celular/efeitos dos fármacos , Melanoma/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Invasividade Neoplásica/patologia , Inibidores de Proteínas Quinases/farmacologia , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/genética , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Células HCT116 , Células HT29 , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Morfolinas/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteína S6 Ribossômica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas , Serina-Treonina Quinases TOR/antagonistas & inibidores , Melanoma Maligno CutâneoRESUMO
Hepatocellular carcinoma (HCC) is the sixth most common cancer, and the third most common cause of cancer related death worldwide. The multi-kinase inhibitor Sorafenib represents the only systemic treatment option until today, and results from clinical trials with allosteric mTOR inhibitors were sobering. Since the PI3K/AKT/mTOR and RAF/MEK/ERK signaling pathways are frequently upregulated in HCC, we have analyzed the effects of AKT inhibitor MK-2206, MEK inhibitor AZD6244 (ARRY 142886) and mTOR kinase inhibitor AZD8055, given as single drugs or in combination, on proliferation and apoptosis of three HCC cell lines in vitro. We show that all three inhibitor combinations synergistically inhibit proliferation of the three HCC cell lines, with the strongest synergistic effect observed after vertical inhibition of AKT and mTORC1/2. We demonstrate that AKT kinase activity is restored 24h after blockade of mTORC1/2 by increased phosphorylation of T308, providing a rationale for combined targeting of AKT and mTOR inhibition in HCC. Our data suggest that a combination of inhibitors targeting those respective pathways may be a viable approach for future application in patients with hepatocellular carcinoma.
RESUMO
PURPOSE: The ABO gene locus is associated with the risk of developing pancreatic ductal adenocarcinoma (PDAC) resulting in an increased incidence in individuals with non-O blood groups. Up to 90% of PDAC specimens display alterations in mucin type O-GalNAc glycosylation. Because aberrant O-GalNAc glycans (Tn and T antigen) are structurally related to blood group A and B glycans, we investigated the role of IgM isoagglutinins in PDAC. EXPERIMENTAL DESIGN: Binding studies of IgM isoagglutinins toward blood group A, B, Tn antigen, and T antigen glycoconjugates from patients with PDAC and healthy individuals were conducted. Isoagglutinin titers and total IgM were compared between patients with PDAC and control group. An anti-A antibody was used for immunoprecipitation of aberrant O-glycosylated tumor proteins and subsequent mass spectromic analysis. RESULTS: We found that IgM isoagglutinins bind blood group antigens, Tn and T glycoconjugates as well as tumor-derived glycoproteins. Blood group A isoagglutinins exhibited a strong binding toward blood group B antigen and T antigen, whereas blood group B showed binding to blood group A antigen and Tn antigen. Furthermore, we confirmed a decreased frequency in individuals with blood group O and observed a significant decrease of IgM isoagglutinin titers in PDAC sera compared with control sera, whereas total IgM levels were unaltered. We identified new PDAC-derived O-GalNAc glycoproteins by mass spectrometry using a blood group A-specific antibody. CONCLUSION: Our data elucidated a novel interaction of blood group IgM isoagglutinins and PDAC O-GalNAc glycoproteins that may contribute to the pathogenesis and progression of pancreatic cancer.
Assuntos
Sistema ABO de Grupos Sanguíneos , Aglutininas/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Imunoglobulina M/metabolismo , Neoplasias Pancreáticas/metabolismo , Polissacarídeos/metabolismo , Sistema ABO de Grupos Sanguíneos/imunologia , Adulto , Idoso , Aglutininas/sangue , Aglutininas/imunologia , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Estudos de Casos e Controles , Feminino , Humanos , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Ligação Proteica , Adulto JovemRESUMO
The phosphoinositide 3-kinase (PI3K) is frequently activated in human cancer cells due to gain of function mutations in the catalytic (p110) and the regulatory (p85) subunits. The regulatory subunit consists of an SH3 domain and two SH2 domains. An oncogenic form of p85α named p65 lacking the c-terminal SH2 domain (cSH2) has been cloned from an irradiation-induced murine thymic lymphoma and transgenic mice expressing p65 in T lymphocytes develop a lymphoproliferative disorder. We have recently detected a c-terminal truncated form of p85α named p76α in a human lymphoma cell line lacking most of the cSH2 domain due to a frame shift mutation. Here, we report that the deletion of the cSH2 domain enhances the activating effects of the n-terminal SH2 domain (nSH2) mutants K379E and R340E on the PI3K/Akt pathway and micro tumor formation in a focus assay. Further analysis revealed that this transforming effect is mediated by activation of the catalytic PI3K isoform p110α and downstream signaling through mTOR. Our data further support a mechanistic model in which mutations of the cSH2 domain of p85α can abrogate its negative regulatory function on PI3K activity via the nSH2 domain of p85α.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/genética , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Deleção de Sequência , Domínios de Homologia de src , Animais , Linhagem Celular , Transformação Celular Neoplásica/metabolismo , Embrião de Galinha , Classe Ia de Fosfatidilinositol 3-Quinase/química , Ativação Enzimática , Fibroblastos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Constitutive activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling has been observed in up to 70% of acute myeloid leukemia. Class I(A) PI3K consists of a catalytic subunit (p110α, p110ß, p110δ) and an adapter subunit (p85α, p55α, p50α, p85ß, p55γ). The p85α adapter subunit stabilizes the catalytic p110 subunit and recruits p110 to the plasma membrane. In addition, p85α inhibits the basal activity of p110α and can negatively regulate signal transduction, as shown for insulin and GM-CSF receptor signaling. Here, we describe that the expression of p85α is posttranscriptionally regulated in several human and murine leukemia cell lines and in a Hodgkin lymphoma cell line (CO) by translational repression. A detailed analysis of CO cells revealed that both wild type and a mutated p85α mRNA are detectable at similar ratios in the nucleus and polysomes. However, while the mutated p85α protein is expressed in CO cells, translation of the wild type p85α mRNA is completely inhibited. Ectopic expression of wild type p85α from a retroviral vector is suppressed in CO cells and in five out of six leukemia cell lines. Our data indicate that leukemia cells can regulate the expression of p85α by posttranscriptional regulation.