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BACKGROUND: Melanoma progression is based on a close interaction between cancer cells and immune cells in the tumor microenvironment (TME). Thus, a better understanding of the mechanisms controlling TME dynamics and composition will help improve the management of this dismal disease. Work from our and other groups has reported the requirement of an active Hedgehog-GLI (HH-GLI) signaling for melanoma growth and stemness. However, the role of the downstream GLI1 transcription factor in melanoma TME remains largely unexplored. METHODS: The immune-modulatory activity of GLI1 was evaluated in a syngeneic B16F10 melanoma mouse model assessing immune populations by flow cytometry. Murine polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were differentiated from bone marrow cells and their immunosuppressive ability was assessed by inhibition of T cells. Conditioned media (CM) from GLI1-overexpressing mouse melanoma cells was used to culture PMN-MDSCs, and the effects of CM were evaluated by Transwell invasion assay and T cell inhibition. Cytokine array analysis, qPCR and chromatin immunoprecipitation were performed to explore the regulation of CX3CL1 expression by GLI1. Human monocyte-derived dendritic cells (moDCs) were cultured in CM from GLI1-silenced patient-derived melanoma cells to assess their activation and recruitment. Blocking antibodies anti-CX3CL1, anti-CCL7 and anti-CXCL8 were used for in vitro functional assays. RESULTS: Melanoma cell-intrinsic activation of GLI1 promotes changes in the infiltration of immune cells, leading to accumulation of immunosuppressive PMN-MDSCs and regulatory T cells, and to decreased infiltration of dendric cells (DCs), CD8 + and CD4 + T cells in the TME. In addition, we show that ectopic expression of GLI1 in melanoma cells enables PMN-MDSC expansion and recruitment, and increases their ability to inhibit T cells. The chemokine CX3CL1, a direct transcriptional target of GLI1, contributes to PMN-MDSC expansion and recruitment. Finally, silencing of GLI1 in patient-derived melanoma cells promotes the activation of human monocyte-derived dendritic cells (moDCs), increasing cytoskeleton remodeling and invasion ability. This phenotype is partially prevented by blocking the chemokine CCL7, but not CXCL8. CONCLUSION: Our findings highlight the relevance of tumor-derived GLI1 in promoting an immune-suppressive TME, which allows melanoma cells to evade the immune system, and pave the way for the design of new combination treatments targeting GLI1.
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Melanoma , Células Supresoras de Origen Mieloide , Microambiente Tumoral , Proteína con Dedos de Zinc GLI1 , Animales , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Ratones , Humanos , Células Supresoras de Origen Mieloide/metabolismo , Células Supresoras de Origen Mieloide/inmunología , Melanoma/patología , Melanoma/metabolismo , Melanoma/inmunología , Melanoma/genética , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/metabolismo , Línea Celular Tumoral , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ratones Endogámicos C57BLRESUMEN
Aberrant activation of Hedgehog (HH) signaling in cancer is the result of genetic alterations of upstream pathway components (canonical) or other oncogenic mechanisms (noncanonical), that ultimately concur to activate the zinc-finger transcription factors GLI1 and GLI2. Therefore, inhibition of GLI activity is a good therapeutic option to suppress both canonical and noncanonical activation of the HH pathway. However, only a few GLI inhibitors are available, and none of them have the profile required for clinical development due to poor metabolic stability and aqueous solubility, and high hydrophobicity. Two promising quinoline inhibitors of GLI were selected by virtual screening and subjected to hit-to-lead optimization, thus leading to the identification of the 4-methoxy-8-hydroxyquinoline derivative JC19. This molecule impaired GLI1 and GLI2 activities in several cellular models interfering with the binding of GLI1 and GLI2 to DNA. JC19 suppressed cancer cell proliferation by enhancing apoptosis, inducing a strong anti-tumor response in several cancer cell lines in vitro. Specificity towards GLI1 and GLI2 was demonstrated by lower activity of JC19 in GLI1- or GLI2-depleted cancer cells. JC19 showed excellent metabolic stability and high passive permeability. Notably, JC19 inhibited GLI1-dependent melanoma xenograft growth in vivo, with no evidence of toxic effects in mice. These results highlight the potential of JC19 as a novel anti-cancer agent targeting GLI1 and GLI2.
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Neoplasias , Proteína con Dedos de Zinc GLI1 , Proteína Gli2 con Dedos de Zinc , Animales , Humanos , Ratones , Proteínas Hedgehog/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteína con Dedos de Zinc GLI1/antagonistas & inhibidores , Proteína Gli2 con Dedos de Zinc/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/patologíaRESUMEN
Desferoxamine (DFO) is currently the golden standard chelator for 89Zr4+, a promising nuclide for positron emission tomography imaging (PET). The natural siderophore DFO had previously been conjugated with fluorophores to obtain Fe(III) sensing molecules. In this study, a fluorescent coumarin derivative of DFO (DFOC) has been prepared and characterized (potentiometry, UV-Vis spectroscopy) for what concerns its protonation and metal coordination properties towards PET-relevant ions (Cu(II), Zr(IV)), evidencing strong similarity with pristine DFO. Retention of DFOC fluorescence emission upon metal binding has been checked (fluorescence spectrophotometry), as it would - and does - allow for optical (fluorescent) imaging, thus unlocking bimodal (PET/fluorescence) imaging for 89Zr(IV) tracers. Crystal violet and MTT assays on NIH-3 T3 fibroblasts and MDA-MB 231 mammary adenocarcinoma cell lines demonstrated, respectively, no cytotoxicity nor metabolic impairment at usual radiodiagnostic concentrations of ZrDFOC. Clonogenic colony-forming assay performed on X-irradiated MDA-MB 231 cells showed no interference of ZrDFOC with radiosensitivity. Morphological biodistribution (confocal fluorescence, transmission electron microscopy) assays on the same cells suggested internalization of the complex through endocytosis. Overall, these results support fluorophore-tagged DFO as a suitable option to achieve dual imaging (PET/fluorescence) probes based on 89Zr.
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Deferoxamina , Radioisótopos , Deferoxamina/química , Radioisótopos/química , Distribución Tisular , Compuestos Férricos , Fluorescencia , Tomografía de Emisión de Positrones/métodos , Quelantes/química , Cumarinas , Línea Celular TumoralRESUMEN
Cholangiocarcinoma (CCA) is characterized by resistance to chemotherapy and a poor prognosis. Therefore, treatments that can effectively suppress tumor growth are urgently needed. Aberrant activation of hedgehog (HH) signaling has been implicated in several cancers, including those of the hepatobiliary tract. However, the role of HH signaling in intrahepatic CCA (iCCA) has not been completely elucidated. In this study, we addressed the function of the main transducer Smoothened (SMO) and the transcription factors (TFs) GLI1 and GLI2 in iCCA. In addition, we evaluated the potential benefits of the combined inhibition of SMO and the DNA damage kinase WEE1. Transcriptomic analysis of 152 human iCCA samples showed increased expression of GLI1, GLI2, and Patched 1 (PTCH1) in tumor tissues compared with nontumor tissues. Genetic silencing of SMO, GLI1, and GLI2 inhibited the growth, survival, invasiveness, and self-renewal of iCCA cells. Pharmacologic inhibition of SMO reduced iCCA growth and viability in vitro, by inducing double-strand break DNA damage, leading to mitotic arrest and apoptotic cell death. Importantly, SMO inhibition resulted in the activation of the G2-M checkpoint and DNA damage kinase WEE1, increasing the vulnerability to WEE1 inhibition. Hence, the combination of MRT-92 with the WEE1 inhibitor AZD-1775 showed increased antitumor activity in vitro and in iCCA xenografts compared with single treatments. These data indicate that combined inhibition of SMO and WEE1 reduces tumor burden and may represent a strategy for the clinical development of novel therapeutic approaches in iCCA.
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Colangiocarcinoma , Proteínas Hedgehog , Humanos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Daño del ADN , Proteínas Tirosina Quinasas/genética , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
Cholangiocarcinoma (CCA) is a poorly treatable type of cancer and its incidence is dramatically increasing. The lack of understanding of the biology of this tumor has slowed down the identification of novel targets and the development of effective treatments. Based on next generation sequencing profiling, alterations in DNA damage response (DDR)-related genes are paving the way for DDR-targeting strategies in CCA. Based on the notion of synthetic lethality, several DDR-inhibitors (DDRi) have been developed with the aim of accumulating enough DNA damage to induce cell death in tumor cells. Observing that DDRi alone could be insufficient for clinical use in CCA patients, the combination of DNA-damaging regimens with targeted approaches has started to be considered, as evidenced by many emerging clinical trials. Hence, novel therapeutic strategies combining DDRi with patient-specific targeted drugs could be the next level for treating cholangiocarcinoma.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/genética , Conductos Biliares Intrahepáticos , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/genética , Daño del ADN , Reparación del ADN , HumanosRESUMEN
Cholangiocarcinoma (CCA) is a poorly treatable type of cancer and, along with hepatocellular carcinoma (HCC), is the predominant type of primitive liver cancer in adults. The lack of understanding of CCA biology has slowed down the identification of novel targets and the development of effective treatments. While tumors share some general characteristics, detailed knowledge of specific features is essential for the development of effectively tailored therapeutic approaches. The Hedgehog (HH) signaling cascade regulates stemness biology, embryonal development, tissue homeostasis, and cell proliferation and differentiation. Its aberrant activation has been associated with a variety of solid and hematological human malignancies. Several HH-inhibiting compounds have been indeed developed as potential anticancer agents in different types of tumors, with Smoothened and GLI inhibitors showing the most promising results. Beside its well-established function in other tumors, findings regarding the HH signaling in CCA are still controversial. Here we will give an overview of the most important clinical and molecular features of cholangiocarcinoma, and we will discuss the available evidence of the crosstalk between the HH signaling pathway and the cholangiocarcinoma cell biology.
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Understanding the molecular events controlling melanoma progression is of paramount importance for the development of alternative treatment options for this devastating disease. Here we report a mechanism regulated by the oncogenic SOX2-GLI1 transcriptional complex driving melanoma invasion through the induction of the sialyltransferase ST3GAL1. Using in vitro and in vivo studies, we demonstrate that ST3GAL1 drives melanoma metastasis. Silencing of this enzyme suppresses melanoma invasion and significantly reduces the ability of aggressive melanoma cells to enter the blood stream, colonize distal organs, seed and survive in the metastatic environment. Analysis of glycosylated proteins reveals that the receptor tyrosine kinase AXL is a major effector of ST3GAL1 pro-invasive function. ST3GAL1 induces AXL dimerization and activation that, in turn, promotes melanoma invasion. Our data support a key role of the ST3GAL1-AXL axis as driver of melanoma metastasis, and highlight the therapeutic potential of targeting this axis to treat metastatic melanoma.