RESUMO
Hepatocyte growth factor (HGF), the ligand for the MET receptor tyrosine kinase, is a tumor-promoting factor that is abundant in the tumor microenvironment. Proteolytic activation of inactive pro-HGF by one or more of the serine endopeptidases matriptase, hepsin, and HGF activator is the rate-limiting step in HGF/MET signaling. Herein, we have rationally designed a novel class of side chain cyclized macrocyclic peptide inhibitors. The new series of cyclic tripeptides has superior metabolic stability and significantly improved pharmacokinetics in mice relative to the corresponding linear peptides. We identified the lead compound VD2173 that potently inhibits matriptase and hepsin, which was tested in parallel alongside the acyclic inhibitor ZFH7116 using both in vitro and in vivo models of lung cancer. We demonstrated that both compounds block pro-HGF activation, abrogate HGF-mediated wound healing, and overcome resistance to EGFR- and MET-targeted therapy in lung cancer models. Furthermore, VD2173 inhibited HGF-dependent growth of lung cancer tumors in mice.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Pulmonares/patologia , Compostos Macrocíclicos/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Progressão da Doença , Descoberta de Drogas , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Compostos Macrocíclicos/sangue , Compostos Macrocíclicos/química , Compostos Macrocíclicos/uso terapêutico , Camundongos , Inibidores de Serina Proteinase/sangue , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/uso terapêutico , Relação Estrutura-AtividadeRESUMO
The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.
Assuntos
Benzotiazóis/farmacologia , Tratamento Farmacológico da COVID-19 , Oligopeptídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/genética , Animais , Benzamidinas/química , Benzotiazóis/farmacocinética , COVID-19/genética , COVID-19/virologia , Linhagem Celular , Desenho de Fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Ésteres/química , Guanidinas/química , Humanos , Pulmão/efeitos dos fármacos , Pulmão/virologia , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Oligopeptídeos/farmacocinética , SARS-CoV-2/patogenicidade , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/ultraestrutura , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacosRESUMO
The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitors with significantly improved activity over existing irreversible inhibitors Camostat and Nafamostat. Lead compound MM3122 ( 4 ) has an IC 50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC 50 of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV SARS-CoV-2 chimeric virus, and an EC 50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East Respiratory Syndrome Coronavirus (MERS-CoV) cell entry with an EC 50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice with a half-life of 8.6 hours in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.
RESUMO
Conjugation of bioactive targeting molecules to nano- or micrometer-sized drug carriers is a pivotal strategy to improve their therapeutic efficiency. Herein, we developed pH- and redox-sensitive hydrogel particles with a surface-conjugated cancer cell targeting ligand for specific tumor-targeting and controlled release of the anticancer drug doxorubicin. The poly(methacrylic acid) (PMAA) hydrogel cubes of 700 nm and 2 µm with a hepsin-targeting (IPLVVPL) surface peptide are produced through multilayer polymer assembly on sacrificial cubical mesoporous cores. Direct peptide conjugation to the disulfide-stabilized hydrogels through a thiol-amine reaction does not compromise the structural integrity, hydrophilicity, stability in serum, or pH/redox sensitivity but does affect internalization by cancer cells. The cell uptake kinetics and the ultimate extent of internalization are controlled by the cell type and hydrogel size. The peptide modification significantly promotes the uptake of the 700 nm hydrogels by hepsin-positive MCF-7 cells due to ligand-receptor recognition but has a negligible effect on the uptake of 2 µm PMAA hydrogels. The selectivity of 700 nm IPLVVPL-PMAA hydrogel cubes to hepsin-overexpressing tumor cells is further confirmed by a 3-10-fold higher particle internalization by hepsin-positive MCF-7 and SK-OV-3 compared to that of hepsin-negative PC-3 cells. This work provides a facile method to fabricate enhanced tumor-targeting carriers of submicrometer size and improves the general understanding of particle design parameters for targeted drug delivery.
Assuntos
Doxorrubicina/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Hidrogéis/química , Neoplasias/tratamento farmacológico , Fragmentos de Peptídeos/química , Ácidos Polimetacrílicos/química , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/química , Sobrevivência Celular , Doxorrubicina/administração & dosagem , Humanos , Neoplasias/patologia , Fragmentos de Peptídeos/metabolismo , Polímeros/química , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Células Tumorais CultivadasRESUMO
Targeted therapeutic agents, such as inhibitors of epithelial growth factor receptor (EGFR), have transformed the management of non-small cell lung cancer (NSCLC) patients. MET-amplified NSCLC cells display resistance to EGFR-targeting agents, but are addicted to MET signaling for survival and proliferation and are sensitive to MET inhibition. However, responsive cancer cells invariably develop resistance to MET-targeted treatment. The tumor microenvironment plays a major role in resistance to anticancer therapy. We demonstrated that fibroblasts block the response of MET-amplified NSCLC cells to the MET kinase inhibitor, JNJ38877605 in an HGF-dependent manner. Thus, MET-amplified NSCLC cells become addicted to HGF upon pharmacological inhibition of MET. HGF restored phosphorylation of MET, EGFR and RON, and maintained pro-survival AKT and ERK signaling in MET-inhibited cells. We developed a small molecule inhibitor of pro-HGF activation, SRI31215, which acts as a triplex inhibitor of the pro-HGF activating proteases matriptase, hepsin and HGF activator (HGFA). SRI31215 blocked crosstalk between tumor cells and fibroblasts and overcame fibroblast-mediated resistance to MET inhibition by preventing fibroblast-mediated reactivation of AKT and ERK signaling. Structurally unrelated triplex inhibitors of matriptase, hepsin and HGFA that we developed in parallel showed similar biological activity. Our data suggest that simultaneous inhibition of HGF and MET is required to overcome resistance to MET inhibitors in MET-amplified NSCLC cells. This provides a rationale for the development of novel combination therapeutic strategies for the treatment of NSCLC patients with MET amplification.
RESUMO
The tumor microenvironment plays a key role in tumor development and progression. Stromal cells secrete growth factors, cytokines and extracellular matrix proteins which promote growth, survival and metastatic spread of cancer cells. Fibroblasts are the predominant constituent of the tumor stroma and Hepatocyte Growth Factor (HGF), the specific ligand for the tyrosine kinase receptor c-MET, is a major component of their secretome. Indeed, cancer-associated fibroblasts have been shown to promote growth, survival and migration of cancer cells in an HGF-dependent manner. Fibroblasts also confer resistance to anti-cancer therapy through HGF-induced epithelial mesenchymal transition (EMT) and activation of pro-survival signaling pathways such as ERK and AKT in tumor cells. Constitutive HGF/MET signaling in cancer cells is associated with increased tumor aggressiveness and predicts poor outcome in cancer patients. Due to its role in tumor progression and therapeutic resistance, both HGF and MET have emerged as valid therapeutic targets. Several inhibitors of MET and HGF are currently being tested in clinical trials. Preclinical data provide a strong indication that inhibitors of HGF/MET signaling overcome both primary and acquired resistance to EGFR, HER2, and BRAF targeting agents. These findings support the notion that co-targeting of cancer cells and stromal cells is required to prevent therapeutic resistance and to increase the overall survival rate of cancer patients. HGF dependence has emerged as a hallmark of therapeutic resistance, suggesting that inhibitors of biological activity of HGF should be included into therapeutic regimens of cancer patients.
RESUMO
The luciferase (LUC) reporter assay is commonly used to study gene expression at the transcriptional level. It is convenient, fast, sensitive, inexpensive, and provides quantitative data about small changes in transcription. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that plays a crucial role in signaling by interferons (IFNs). Here, we describe LUC reporter studies that address the role of histone deacetylase (HDAC) activity in STAT1-dependent gene activation. These experiments include overexpression of HDAC1, HDAC2, HDAC3, and HDAC4 as well as silencing of HDAC1, HDAC2, and HDAC3 through RNA interference in mammalian cancer cells.
Assuntos
Bioensaio , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Inibidores de Histona Desacetilases/farmacologia , Fator de Transcrição STAT1/genética , Transcrição Gênica/efeitos dos fármacos , Acetilação , Ácido Butírico/farmacologia , Linhagem Celular Tumoral , Epigênese Genética , Genes Reporter , Células HCT116 , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Interferon gama/farmacologia , Luciferases/genética , Luciferases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT1/metabolismoRESUMO
The binding of hepatocyte growth factor (HGF) to its receptor MET activates a signaling cascade that promotes cell survival, proliferation, cell scattering, migration and invasion of malignant cells. HGF is secreted by cancer cells or by tumor-associated fibroblasts as pro-HGF, an inactive precursor. A key step in the regulation of HGF/MET signaling is proteolytic processing of pro-HGF to its active form by one of the three serine proteases, matriptase, hepsin or HGF activator (HGFA).We developed SRI 31215, a small molecule that acts as a triplex inhibitor of matriptase, hepsin and HGFA and mimics the activity of HAI-1/2, endogenous inhibitors of HGF activation. We demonstrated that SRI 31215 inhibits fibroblast-induced MET activation, epithelial-mesenchymal transition and migration of cancer cells. SRI 31215 overcomes primary resistance to cetuximab and gefitinib in HGF-producing colon cancer cells and prevents fibroblast-mediated resistance to EGFR inhibitors. Thus, SRI 31215 blocks signaling between cancer cells and fibroblasts and inhibits the tumor-promoting activity of cancer-associated fibroblasts.Aberrant HGF/MET signaling supports cell survival, proliferation, angiogenesis, invasion and metastatic spread of cancer cells, establishing HGF and MET as valid therapeutic targets. Our data demonstrate that inhibitors of HGF activation, such as SRI 31215, merit investigation as potential therapeutics in tumors that are addicted to HGF/MET signaling. The findings reported here also indicate that inhibitors of HGF activation overcome primary and acquired resistance to anti-EGFR therapy, providing a rationale for concurrent inhibition of EGFR and HGF to prevent therapeutic resistance and to improve the outcome of cancer patients.
Assuntos
Antineoplásicos/farmacologia , Benzamidinas/farmacologia , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Precursores de Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Pirimidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fibroblastos Associados a Câncer/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Precursores de Proteínas/efeitos dos fármacosRESUMO
In this letter we report first nonpeptide inhibitors of hepatocyte growth factor (HGF) activation. These compounds inhibit the three proteases (matriptase, hepsin, and HGF activator) required for HGF maturation. We show that 6, 8a, 8b, and 8d block activation of fibroblast-derived pro-HGF, thus preventing fibroblast-induced scattering of DU145 prostate cancer cells. Compound 6 (SRI 31215) is very soluble (91 µM) and has excellent microsome stability (human t 1/2 = 162 min; mouse t 1/2 = 296 min). In mouse 6 has an in vivo t 1/2 = 5.8 h following IV administration. The high solubility of 6 and IV t 1/2 make this compound a suitable prototype "triplex inhibitor" for the study of the inhibition of HGF activation in vivo.
RESUMO
Upregulation of the HGF and MSP growth-factor processing serine endopeptidases HGFA, matriptase and hepsin is correlated with increased metastasis in multiple tumor types driven by c-MET or RON kinase signaling. We rationally designed P1' α-ketobenzothiazole mechanism-based inhibitors of these proteases. Structure-activity studies are presented, which resulted in the identification of potent inhibitors with differential selectivity. The tetrapeptide inhibitors span the P1-P1' substrate cleavage site via a P1' amide linker off the benzothiazole, occupying the S3' pocket. Optimized inhibitors display sub-nanomolar enzyme inhibition against one, two, or all three of HGFA, matriptase, and hepsin. Several compounds also have good selectivity against the related trypsin-like proteases, thrombin and Factor Xa. Finally, we show that inhibitors block the fibroblast (HGF)-mediated migration of invasive DU145 prostate cancer cells. In addition to prostate cancer, breast, colon, lung, pancreas, gliomas, and multiple myeloma tumors all depend on HGF and MSP for tumor survival and progression. Therefore, these unique inhibitors have potential as new therapeutics for a diverse set of tumor types.
Assuntos
Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Oligopeptídeos/farmacologia , Inibidores de Serina Proteinase/farmacologia , Antineoplásicos/síntese química , Benzotiazóis/síntese química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Fator Xa/metabolismo , Humanos , Modelos Moleculares , Oligopeptídeos/síntese química , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/síntese química , Transdução de Sinais , Relação Estrutura-Atividade , Trombina/metabolismoRESUMO
Colon cancer development and malignant progression are driven by genetic and epigenetic alterations in tumor cells and by factors from the tumor microenvironment. Cancer cells become reliant on the activity of specific oncogenes and on prosurvival and proliferative signals they receive from the abnormal environment they create and reside in. Accordingly, the response to anticancer therapy is determined by genetic and epigenetic changes that are intrinsic to tumor cells and by the factors present in the tumor microenvironment. Recent advances in the understanding of the involvement of the tumor microenvironment in tumor progression and therapeutic response are optimizing the application of prognostic and predictive factors in colon cancer. Moreover, new targets in the tumor microenvironment that are amenable to therapeutic intervention have been identified. Because stromal cells are with rare exceptions genetically stable, the tumor microenvironment has emerged as a preferred target for therapeutic drugs. In this review, we discuss the role of stromal fibroblasts and macrophages in colon cancer progression and in the response of colon cancer patients to therapy.
RESUMO
Activating Ras mutations, present in about 20% of human cancers, compromise the GTPase activity of Ras and therefore trigger accumulation of Ras in the GTP-bound state. Among the three family members, K-Ras, H-Ras and N-Ras, K-Ras is the most frequently mutated gene, with 30-50% of colon cancer patients harboring activating K-Ras mutations. Oncogenic mutations of K-Ras have been found at codons 12, 13, 61 and 146. Activation of Ras triggers constitutive activation of signaling pathways, including the MAPK and AKT pathways, which allows tumor cells to proliferate in the absence of growth factors and increases their survival. In addition, activated Ras triggers inflammation and thus promotes tumor progression in a cell non-autonomous manner. The presence of K-Ras mutations not only has prognostic value, but it also predicts the responsiveness of colon cancer patients to inhibitors of EGFR signaling.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/genética , Genes ras/genética , Animais , Proliferação de Células/genética , Sobrevivência Celular/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Progressão da Doença , Receptores ErbB/antagonistas & inibidores , Homeostase , Humanos , Mutação , Prognóstico , Transdução de Sinais/genéticaRESUMO
Calcitriol, 1α, 25-dihydroxyvitamin D3 (1,25 (OH)2D3), the most active form of vitamin D, is a pleotropic hormone with a wide range of biological activities. Due to its ability to regulate calcium and phosphate metabolism, 1,25D3 plays a major role in bone health. In addition, 1,25D3 binds to the vitamin D receptor and thereby regulates the expression of a number of genes which control growth, differentiation and survival of cancer cells. In agreement, the levels of vitamin D3 appear to be an essential determinant for the development and progression of colon cancer and supplementation with vitamin D3 is effective in suppressing intestinal tumorigenesis in animal models. Vitamin D3 has been estimated to lower the incidence of colorectal cancer by 50%, which is consistent with the inverse correlation between dietary vitamin D3 intake or sunlight exposure and human colorectal cancer. Several studies confirmed that increasing vitamin D3 lowers colon cancer incidence, reduces polyp recurrence, and that sufficient levels of vitamin D3 are associated with better overall survival of colon cancer patients. Vitamin D regulates the homeostasis of intestinal epithelium by modulating the oncogenic Wnt signaling pathway and by inhibiting tumor-promoting inflammation. Both activities contribute to the ability of 1,25D3 to prevent the development and progression of colon cancer.
RESUMO
Reovirus is a double stranded RNA virus, with an intrinsic preference for replication in KRAS mutant cells. As 45% of human colorectal cancers (CRC) harbor KRAS mutations, we sought to investigate its efficacy in KRAS mutant CRC cells, and examine its impact in combination with the topoisimerase-1 inhibitor, irinotecan. Reovirus efficacy was examined in the KRAS mutant HCT116, and the isogenic KRAS WT Hke3 cell line, and in the non-malignant rat intestinal epithelial cell line. Apoptosis was determined by flow cytometry and TUNEL staining. Combination treatment with reovirus and irintoecan was investigated in 15 CRC cell lines, including the HCT116 p21 isogenic cell lines. Reovirus preferentially induced apoptosis in KRAS mutant HCT116 cells compared to its isogenic KRAS WT derivative, and in KRAS mutant IEC cells. Reovirus showed a greater degree of caspase 3 activation with PARP 1 cleavage, and preferential inhibition of p21 protein expression in KRAS mutant cells. Reovirus synergistically induced growth inhibition when combined with irinotecan. This synergy was lost upon p21 gene knock out. Reovirus preferentially induces apoptosis in KRAS mutant colon cancer cells. Reovirus and irinotecan combination therapy is synergistic, p21 mediated, and represents a novel potential treatment for patients with CRC.
Assuntos
Antineoplásicos/uso terapêutico , Camptotecina/análogos & derivados , Neoplasias Colorretais/terapia , Mutação/genética , Terapia Viral Oncolítica , Proteínas Proto-Oncogênicas/genética , Reoviridae/fisiologia , Proteínas ras/genética , Animais , Apoptose , Western Blotting , Camptotecina/uso terapêutico , Ciclo Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Terapia Combinada , Sinergismo Farmacológico , Humanos , Irinotecano , Microscopia Eletrônica de Varredura , Proteínas Proto-Oncogênicas p21(ras) , Ratos , Células Tumorais CultivadasRESUMO
Signaling between tumor cells and the associated stroma has an important impact on cancer initiation and progression. The tumor microenvironment has a paradoxical role in tumor progression and fibroblasts, a major component of the tumor stroma, have been shown to either inhibit or promote cancer development. In this study, we established that normal intestinal fibroblasts activate STAT1 signaling in colon cancer cells and, in contrast to cancer-associated fibroblasts, inhibit growth of tumor cells. Treatment of 18Co fibroblasts with the proinflammatory cytokine TNFα interfered with their ability to trigger STAT1 signaling in cancer cells. Accordingly, intestinal myofibroblasts isolated from patients with ulcerative colitis or Crohn's disease, which are activated and produce high levels of TNFα, failed to stimulate STAT1 signaling in tumor cells, demonstrating that activated myofibroblasts lose the ability to trigger growth-inhibitory STAT1 signaling in tumor cells. Finally, we confirmed that silencing of STAT1 in tumor cells alters the crosstalk between tumor cells and fibroblasts, suggesting STAT1 as a novel link between intestinal inflammation and colon cancer. We demonstrated that normal fibroblasts restrain the growth of carcinoma cells, at least in part, through the induction of STAT1 signaling in cancer cells and showed that changes in the microenvironment, as they occur in inflammatory bowel disease, alter the crosstalk between carcinoma cells and fibroblasts, perturb the homeostasis of intestinal tissue, and thereby contribute to tumor progression.
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BACKGROUND: Tumor cells become addicted to both activated oncogenes and to proliferative and pro-survival signals provided by the abnormal tumor microenvironment. Although numerous soluble factors have been identified that shape the crosstalk between tumor cells and stroma, it has not been established how oncogenic mutations in the tumor cells alter their interaction with normal cells in the tumor microenvironment. PRINCIPAL FINDINGS: We showed that the isogenic HCT116 and Hke-3 cells, which differ only by the presence of the mutant kRas allele, both stimulate macrophages to produce IL1ß. In turn, macrophages enhanced Wnt signaling, proliferation and survival in both HCT116 and Hke-3 cells, demonstrating that signaling by oncogenic kRas in tumor cells does not impact their interaction with macrophages. HCT116 cells are heterozygous for ß-catenin (HCT116(WT/MT)), harboring one wild type (WT) and one mutant (MT) allele, but isogenic lines that carry only the WT (HCT116(WT)) or MT ß-catenin allele (HCT116(MT)) have been generated. We showed that macrophages promoted Wnt signaling in cells that carry the MT ß-catenin allele, but not in HCT116(WT) cells. Consistent with this observation, macrophages and IL1ß failed to stabilize Snail in HCT116(WT) cells, and to protect these cells from TRAIL-induced apoptosis. Finally, we demonstrated that HCT116 cells expressing dominant negative TCF4 (dnTCF4) or HCT116 cells with silenced Snail failed to stimulate IL1ß production in macrophages, demonstrating that tumor cells activate macrophages via a Wnt-dependent factor. SIGNIFICANCE: Our data demonstrate that oncogenic ß-catenin mutations in tumor cells, and subsequent activation of Wnt signaling, not only trigger cell-intrinsic alterations, but also have a significant impact on the crosstalk of tumor cells with the tumor associated macrophages.
Assuntos
Comunicação Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Macrófagos/metabolismo , Mutação , Fatores de Transcrição/metabolismo , beta Catenina/genética , Alelos , Apoptose , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Genes ras , Células HCT116 , Humanos , Macrófagos/imunologia , Modelos Biológicos , Monócitos/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Proteínas Wnt/metabolismoRESUMO
Patients with inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease, are at increased risk of developing colon cancer, confirming that chronic inflammation predisposes to development of tumors. Moreover, it appears that colon cancers that do not develop as a complication of inflammatory bowel disease are also driven by inflammation, because it has been shown that regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) lowers the mortality from sporadic colon cancer and results in regression of adenomas in familial adenomatous polyposis (FAP) patients, who inherit a mutation in the Apc gene. Colorectal cancer therefore represents a paradigm for the link between inflammation and cancer. Inflammation is driven by soluble factors, cytokines and chemokines, which can be produced by tumor cells themselves or, more often, by the cells recruited to the tumor microenvironment. Inflammatory cytokines and chemokines promote growth of tumor cells, perturb their differentiation, and support the survival of cancer cells. Tumor cells become addicted to inflammatory stroma, suggesting that the tumor microenvironment represents an attractive target for preventive and therapeutic strategies. Proinflammatory cytokines, such as TNFα, IL-6 and IL-1ß, or transcription factors that are required for signaling by these cytokines, including NF-κB and STATs, are indeed emerging as potential targets for anticancer therapy. TNFα antagonists are in phase I/II clinical trials and have been shown to be well tolerated in patients with solid tumors, and IL-1ß antagonists that ameliorate several inflammatory disorders characterized by excessive IL-1ß production, will likely follow. Therefore, development of drugs that normalize the tumor microenvironment or interrupt the crosstalk between the tumor and the tumor microenvironment is an important approach to the management of cancer.
Assuntos
Neoplasias do Colo/metabolismo , Citocinas/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Animais , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Citocinas/imunologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologiaRESUMO
BACKGROUND: We recently reported that colon tumor cells stimulate macrophages to release IL-1beta, which in turn inactivates GSK3beta and enhances Wnt signaling in colon cancer cells, generating a self-amplifying loop that promotes the growth of tumor cells. PRINCIPAL FINDINGS: Here we describe that macrophages protect HCT116 and Hke-3 colon cancer cells from TRAIL-induced apoptosis. Inactivation of IL-1beta by neutralizing IL-1beta antibody, or silencing of IL-1beta in macrophages inhibited their ability to counter TRAIL-induced apoptosis. Accordingly, IL-1beta was sufficient to inhibit TRAIL-induced apoptosis. TRAIL-induced collapse of the mitochondrial membrane potential (Delta psi) and activation of caspases were prevented by macrophages or by recombinant IL-1beta. Pharmacological inhibition of IL-1beta release from macrophages by vitamin D(3), a potent chemopreventive agent for colorectal cancer, restored the ability of TRAIL to induce apoptosis of tumor cells cultured with macrophages. Macrophages and IL-1beta failed to inhibit TRAIL-induced apoptosis in HCT116 cells expressing dnIkappaB, dnAKT or dnTCF4, confirming that they oppose TRAIL-induced cell death through induction of Wnt signaling in tumor cells. We showed that macrophages and IL-1beta stabilized Snail in tumor cells in an NF-kappaB/Wnt dependent manner and that Snail deficient tumor cells were not protected from TRAIL-induced apoptosis by macrophages or by IL-1beta, demonstrating a crucial role of Snail in the resistance of tumor cells to TRAIL. SIGNIFICANCE: We have identified a positive feedback loop between tumor cells and macrophages that propagates the growth and promotes the survival of colon cancer cells: tumor cells stimulate macrophages to secrete IL-1beta, which in turn, promotes Wnt signaling and stabilizes Snail in tumor cells, conferring resistance to TRAIL. Vitamin D(3) halts this amplifying loop by interfering with the release of IL-1beta from macrophages. Accordingly, vitamin D(3) sensitizes tumor cells to TRAIL-induced apoptosis, suggesting that the therapeutic efficacy of TRAIL could be augmented by this readily available chemopreventive agent.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Interleucina-1beta/metabolismo , Macrófagos/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Fatores de Transcrição/metabolismo , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Colecalciferol/farmacologia , Neoplasias do Colo/metabolismo , Imunofluorescência , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Proteínas Wnt/metabolismoRESUMO
Progression of colon cancer from microadenoma to macroscopic tumors is coupled to augmentation of canonical Wnt signaling. We recently reported that tumor associated macrophages, through interleukin 1ß (IL-1ß) dependent phosphorylation of GSK3ß, promote Wnt signaling in colon cancer cells, demonstrating that proinflammatory cytokines can enhance TCF4/ß-catenin transcriptional activity in tumor cells. Here we investigated the pathway whereby IL-1ß inactivates GSK3ß and promotes Wnt signaling in colon cancer cells. We showed that normal human monocytes, THP1 macrophages and IL-1 failed to induce Wnt signaling in tumor cells expressing dominant negative IκB (dnIκB), demonstrating that macrophages and IL-1 activate Wnt signaling in a NF-κB-dependent manner. NF-κB activity was required for macrophages and IL-1 to activate PDK1 and AKT in tumor cells and thereby inhibit GSK3ß activity. Consistently, dominant negative AKT (dnAKT), or pharmacological inhibition of AKT in tumor cells, prevented macrophage/IL-1 mediated phosphorylation of GSK3ß, activation of Wnt signaling, and induction of c-jun and c-myc, confirming that macrophages and IL-1 promote Wnt signaling in an AKT dependent manner. Finally, we showed IL-1 and macrophages failed to promote growth of colon cancer cells with impaired NF-κB or AKT signaling, confirming the requirement for NF-κB and AKT activation for the protumorigenic activity of tumor associated macrophages. Thus, we showed that IL-1 and tumor associated macrophages activate NF-κB-dependent PDK1/AKT signaling in tumor cells, and thereby inactivate GSK3ß, enhance Wnt signaling and promote growth of colon cancer cells, establishing a novel molecular link between inflammation and tumor growth.
RESUMO
Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke-3 cells. However, the presence of oncogenic kRas significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransformed intestinal epithelial cells with inducible expression of kRasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3.