RESUMEN
BACKGROUND: Ceramide is a bioeffector that mediates various cellular processes, including apoptosis. However, the mechanism underlying ceramide function in apoptosis is apparently cell type-dependent and is not well-understood. We aimed at identifying molecular targets of ceramide in metastatic human colon and breast cancer cells, and determining the efficacy of ceramide analog in suppression of colon and breast cancer metastasis. METHODS: The activity of and mechanism underlying ceramide as a cytotoxic agent, and as a sensitizer for Fas-mediated apoptosis was analyzed in human cell lines established from primary or metastatic colon and breast cancers. The efficacy of ceramide analog LCL85 in suppression of metastasis was examined in preclinical mouse tumor models. RESULTS: Exposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to Fas-mediated apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to Fas-mediated apoptosis. LCL85 resembles Smac mimetic BV6 in sensitization of colon carcinoma cells to Fas-mediated apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. LCL85 also decreased xIAP1 and cIAP1 protein levels and sensitized metastatic human breast cancer cells to Fas-mediated apoptosis. Silencing xIAP and cIAP1 with specific siRNAs significantly increased the metastatic human colon carcinoma cell sensitivity to Fas-mediated apoptosis, suggesting that IAP proteins mediate apoptosis resistance in metastatic human colon carcinoma cells and ceramide induces IAP protein degradation to sensitize the tumor cells to apoptosis induction. Consistent with its apoptosis sensitization activity, subtoxic doses of LCL85 suppressed colon carcinoma cell metastatic potential in an experimental lung metastasis mouse model, as well as breast cancer growth and spontaneous lung metastasis in an orthotopic breast cancer mouse model. CONCLUSION: We have identified xIAP and cIAP1 as molecular targets of ceramide and determined that ceramide analog LCL85 is an effective sensitizer in overcoming resistance of human cell lines established from metastatic colon and breast cancers to apoptosis induction to suppress metastasis in vivo.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Ceramidas/farmacología , Neoplasias del Colon/tratamiento farmacológico , Proteínas Inhibidoras de la Apoptosis/metabolismo , Propanolaminas/farmacología , Compuestos de Piridinio/farmacología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/secundario , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/secundario , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Ratones , Ratones Endogámicos BALB C , Terapia Molecular Dirigida , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Transfección , Ubiquitina-Proteína Ligasas , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Receptor fas/metabolismoRESUMEN
Radiation modulates both tumor cells and immune cells in the tumor microenvironment to exert its anti-tumor activity; however, the molecular connection between tumor cells and immune cells that mediates radiation-exerted tumor suppression activity in the tumor microenvironment is largely unknown. We report here that radiation induces rapid activation of the p65/p50 and p50/p50 NF-κB complexes in human soft tissue sarcoma (STS) cells. Radiation-activated p65/p50 and p50/p50 bind to the TNFα promoter to activate its transcription in STS cells. Radiation-induced TNFα induces tumor cell death in an autocrine manner. A sublethal dose of Smac mimetic BV6 induces cIAP1 and cIAP2 degradation to increase tumor cell sensitivity to radiation-induced cell death in vitro and to enhance radiation-mediated suppression of STS xenografts in vivo. Inhibition of caspases, RIP1, or RIP3 blocks radiation/TNFα-induced cell death, whereas inhibition of RIP1 blocks TNFα-induced caspase activation, suggesting that caspases and RIP1 act sequentially to mediate the non-compensatory cell death pathways. Furthermore, we determined in a syngeneic sarcoma mouse model that radiation up-regulates IRF3, IFNß, and the T cell chemokines CCL2 and CCL5 in the tumor microenvironment, which are associated with activation and increased infiltration of Th1/Tc1 T cells in the tumor microenvironment. Moreover, tumor-infiltrating T cells are in their active form since both the perforin and FasL pathways are activated in irradiated tumor tissues. Consequently, combined BV6 and radiation completely suppressed tumor growth in vivo. Therefore, radiation-induced NF-κB functions as a molecular link between tumor cells and immune cells in the tumor microenvironment for radiation-mediated tumor suppression.
Asunto(s)
Neoplasias Colorrectales/prevención & control , Rayos gamma , FN-kappa B/metabolismo , Sarcoma/prevención & control , Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Animales , Apoptosis/efectos de la radiación , Biomarcadores de Tumor/metabolismo , Proliferación Celular/efectos de la radiación , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Sarcoma/inmunología , Sarcoma/metabolismo , Sarcoma/patología , Linfocitos T/efectos de la radiación , Células Tumorales Cultivadas , Microambiente Tumoral/efectos de la radiación , Factor de Necrosis Tumoral alfa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A have been the subject of extensive studies; however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wild-type mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoter to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoter to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer, and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development.