RESUMEN
Inflammation plays a critical role in thyroid cancer onset and progression. We previously characterized the in vitro interplay between macrophages and senescent human thyrocytes and thyroid tumor-derived cell lines, modeling the early and the late thyroid tumor phases, respectively. We reported that both models are able to induce pro-tumoral M2-like macrophage polarization, through the activation of the COX2-PGE2 axis. Here, we investigated the presence of macrophage infiltrating cells in mouse xenografts derived from the above described cells models. We showed that subcutaneous injection in immunodeficient mice of both senescent human thyrocytes and thyroid tumor-derived cell lines elicits macrophage recruitment. Furthermore, considering the type of macrophage infiltrate, we observed a stronger infiltration of Arginase I positive cells (M2-like). Overall, these results demonstrate the in vivo capability of senescent and tumor thyroid cells to recruit and polarize macrophages, suggesting that the promotion of a pro-tumoral microenvironment through tumor associated macrophages may occurs in late as well as in early thyroid tumor stages, favoring tumor onset and progression.
RESUMEN
The coatomer protein complex zeta 1 (COPZ1) represents a non-oncogene addiction for thyroid cancer (TC); its depletion impairs the viability of thyroid tumor cells, leads to abortive autophagy, ER stress, UPR and apoptosis, and reduces tumor growth of TC xenograft models. In this study we investigated the molecular pathways activated by COPZ1 depletion and the paracrine effects on cellular microenvironment and immune response. By comprehensive and target approaches we demonstrated that COPZ1 depletion in TPC-1 and 8505C thyroid tumor cell lines activates type I IFN pathway and viral mimicry responses. The secretome from COPZ1-depleted cells was enriched for several inflammatory molecules and damage-associated molecular patterns (DAMPs). Moreover, we found that dendritic cells, exposed to these secretomes, expressed high levels of differentiation and maturation markers, and stimulated the proliferation of naïve T cells. Interestingly, T cells stimulated with COPZ1-depleted cells showed increased cytotoxic activity against parental tumor cells. Collectively, our findings support the notion that targeting COPZ1 may represent a promising therapeutic approach for TC, considering its specificity for cancer cells, the lack of effect on normal cells, and the capacity to prompt an anti-tumor immune response.
Asunto(s)
Autofagia , Proteína Coatómero/antagonistas & inhibidores , Muerte Celular Inmunogénica , Interferón Tipo I/metabolismo , Linfocitos T/inmunología , Neoplasias de la Tiroides/inmunología , Neoplasias de la Tiroides/patología , Apoptosis , Proliferación Celular , Humanos , Transducción de Señal , Neoplasias de la Tiroides/metabolismo , Células Tumorales CultivadasRESUMEN
Thyroid carcinoma (TC) is the most common malignancy of endocrine organs with an increasing incidence in industrialized countries. The majority of TC are characterized by a good prognosis, even though cases with aggressive forms not cured by standard therapies are also present. Moreover, target therapies have led to low rates of partial response and prompted the emergence of resistance, indicating that new therapies are needed. In this review, we summarize current literature about the non-oncogene addiction (NOA) concept, which indicates that cancer cells, at variance with normal cells, rely on the activity of genes, usually not mutated or aberrantly expressed, essential for coping with the transformed phenotype. We highlight the potential of non-oncogenes as a point of intervention for cancer therapy in general, and present evidence for new putative non-oncogenes that are essential for TC survival and that may constitute attractive new therapeutic targets.
RESUMEN
BACKGROUND: Thyroid carcinoma includes several variants characterized by different biological and clinical features: from indolent microcarcinoma to undifferentiated and aggressive anaplastic carcinoma. Inflammation plays a critical role in thyroid tumors. Conditions predisposing to cancer, as well as oncogene activity, contribute to the construction of an inflammatory microenvironment that facilitates thyroid tumor progression. Moreover, oncogene-induced senescence, a mechanism tightly connected with inflammation, and able to restrain or promote cancer progression, is involved in thyroid cancer. The interactions between thyroid tumor cells and the microenvironment are not completely clarified. METHODS: We characterize in vitro the interplay between macrophages and senescent thyrocytes and tumor-derived cell lines, modeling early and late thyroid tumor stages, respectively. Purified peripheral blood-derived human monocytes were exposed to thyroid cell-derived conditioned medium (CM) and assessed for phenotype by flow cytometry. The factors secreted by thyroid cells and macrophages were identified by gene expression analysis and ELISA. The protumoral effect of macrophages was assessed by wound healing assay on K1 thyroid tumor cells. The expression of PTGS2 and M2 markers in thyroid tumors was investigated in publicly available datasets. RESULTS: Human monocytes exposed to CM from senescent thyrocytes and thyroid tumor cell lines undergo M2-like polarization, showing high CD206 and low MHC II markers, and upregulation of CCL17 secretion. The obtained M2-like macrophages displayed tumor-promoting activity. Among genes overexpressed in polarizing cells, we identified the prostaglandin-endoperoxide synthase enzyme (PTGS2/COX-2), which is involved in the production of prostaglandin E2 (PGE2). By using COX-2 inhibitors we demonstrated that the M2-like polarization ability of thyroid cells is related to the production of PGE2. Co-expression of PTGS2 and M2 markers is observed a significant fraction of human thyroid tumors. CONCLUSIONS: Our results demonstrate that both senescent thyrocytes and thyroid tumor cell lines trigger M2-like macrophage polarization that is related to PGE2 secretion. This suggests that the interaction with the microenvironment occurs at both early and late thyroid tumor stages, and favors tumor progression. The co-expression of PTGS2 gene and M2 markers in human thyroid carcinoma highlights the possibility to counteract tumor growth through COX-2 inhibition.
Asunto(s)
Senescencia Celular/efectos de los fármacos , Ciclooxigenasa 2/genética , Inflamación/tratamiento farmacológico , Neoplasias de la Tiroides/tratamiento farmacológico , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Polaridad Celular/efectos de los fármacos , Senescencia Celular/genética , Quimiocina CCL17/genética , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Inhibidores de la Ciclooxigenasa 2/farmacología , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación/genética , Inflamación/patología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Monocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Epiteliales Tiroideas/efectos de los fármacos , Células Epiteliales Tiroideas/patología , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patologíaRESUMEN
Even if thyroid tumors are generally curable, a fraction will develop resistance to therapy and progress towards undifferentiated forms, whose treatment remains a demanding challenge. To identify potential novel targets for treatment of thyroid cancer, in a previous study using siRNA-mediated functional screening, we identified several genes that are essential for the growth of thyroid tumor, but not normal cells. Among the top-ranking hits, we found microtubule associated serine/threonine kinase-like (MASTL), which is known to play an essential role in mitosis regulation, and is also involved in the DNA damage response. Herein, we examine the effects of MASTL depletion on growth and viability of thyroid tumor cells. MASTL depletion impaired cell proliferation and increased the percentage of cells presenting nuclear anomalies, which are indicative of mitotic catastrophe. Furthermore, MASTL depletion was associated with enhanced DNA damage. All these effects eventually led to cell death, characterized by the presence of apoptotic markers. Moreover, MASTL depletion sensitized thyroid tumor cells to cisplatin. Our results demonstrate that MASTL represents vulnerability for thyroid tumor cells, which could be explored as a therapeutic target for thyroid cancer.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Proteínas Asociadas a Microtúbulos/deficiencia , Mitosis , Proteínas Serina-Treonina Quinasas/deficiencia , Neoplasias de la Tiroides/enzimología , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Histonas/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/genética , Mitosis/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patologíaRESUMEN
Thyroid carcinoma is generally associated with good prognosis, but no effective treatments are currently available for aggressive forms not cured by standard therapy. To find novel therapeutic targets for this tumor type, we had previously performed a siRNA-based functional screening to identify genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same extent for the viability of normal cells (non-oncogene addiction paradigm). Among those, we found the coatomer protein complex ζ1 (COPZ1) gene, which is involved in intracellular traffic, autophagy and lipid homeostasis. In this paper, we investigated the mechanisms through which COPZ1 depletion leads to thyroid tumor cell death. We showed that siRNA-mediated COPZ1 depletion causes abortive autophagy, endoplasmic reticulum stress, unfolded protein response and apoptosis. Interestingly, we observed that mouse tumor xenografts, locally treated with siRNA targeting COPZ1, showed a significant reduction of tumor growth. On the whole, we demonstrated for the first time the crucial role of COPZ1 in the viability of thyroid tumor cells, suggesting that it may be considered an attractive target for novel therapeutic approaches for thyroid cancer.
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Proteína Coatómero/metabolismo , Tratamiento con ARN de Interferencia , Neoplasias de la Tiroides/terapia , Animales , Apoptosis , Autofagia , Línea Celular Tumoral , Supervivencia Celular , Proteína Coatómero/genética , Estrés del Retículo Endoplásmico , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Desnudos , Interferencia de ARN , Transducción de Señal , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Factores de Tiempo , Transfección , Carga Tumoral , Respuesta de Proteína Desplegada , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oncogene-induced senescence (OIS) is a robust and sustained antiproliferative response to oncogenic stress and constitutes an efficient barrier to tumour progression. We have recently proposed that OIS may be involved in the pathogenesis of thyroid carcinoma by restraining tumour progression as well as the transition of well differentiated to more aggressive variants. Here, an OIS inducible model was established and used for dissecting the molecular mechanisms and players regulating senescence in human primary thyrocytes. We show that oncogenic RAS induces senescence in thyrocytes as judged by changes in cell morphology, activation of p16INK4a and p53/p21CIP1 tumour suppressor pathways, senescence-associated ß-galactosidase (SA-ß-Gal) activity, and induction of proinflammatory components including IL-8 and its receptor CXCR2. Using RNA interference (RNAi) we demonstrate that p16INK4a is necessary for the onset of senescence in primary thyrocytes as its depletion rescues RAS-induced senescence. Furthermore, we found that IL-8/CXCR2 network reinforces the growth arrest triggered by oncogenic RAS, as its abrogation is enough to resume proliferation. Importantly, we observed that CXCR2 expression coexists with OIS markers in thyroid tumour samples, suggesting that CXCR2 contributes to senescence, thus limiting thyroid tumour progression.
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Genes ras , Mediadores de Inflamación/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Glándula Tiroides/metabolismo , Proliferación Celular , Forma de la Célula , Células Cultivadas , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación de la Expresión Génica , Humanos , Interleucina-8/metabolismo , Proteína Oncogénica p21(ras)/genética , Cultivo Primario de Células , Interferencia de ARN , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Transducción de Señal , Glándula Tiroides/inmunología , Glándula Tiroides/patología , Factores de Tiempo , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
CONTEXT: Papillary thyroid carcinoma (PTC) is the most frequent thyroid tumor and is responsible for the overall increase in thyroid cancer incidence. S100A11 (calgizzarin), a member of the S100 Ca(2+)-binding protein family, is involved in several different biological processes. S100A11 has been found up-regulated in PTC, both at the mRNA and protein levels. OBJECTIVE: Through a combination of expression analysis and functional in vitro and in vivo studies, we have attempted to gain insight into the relevance of S100A11 overexpression in PTC biology. DESIGN: The expression of the S100A11 gene in PTC was investigated in several gene expression data sets. The effect of S100A11 silencing on the hallmarks of the malignant phenotype of several PTC-derived cell lines was investigated. In NIH3T3 cells, the cooperation of S100A11 with the different PTC-specific oncogenes was assessed. RESULTS: We found that the S100A11 gene expression is frequently up-regulated in PTC, anaplastic thyroid carcinoma, but not in follicular thyroid carcinoma. S100A11 overexpression was also detected in PTC-derived cell lines, which were then used for functional studies. S100A11 silencing in PTC-derived cell lines did not affect cell proliferation, whereas it reduced the loss of contact inhibition, anchorage-independent growth, and resistance to anoikis. Cotransfection experiments in NIH3T3 cells showed that overexpression of the S100A11 gene was able to enhance the transforming capabilities of the different PTC-associated oncogenes by affecting the loss of contact inhibition, anchorage-independent growth, and in vivo tumor formation. CONCLUSION: Our data indicate that S100A11 overexpression exerts a protumoral functional role in PTC pathogenesis.
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Carcinoma Papilar/genética , Proteínas S100/genética , Neoplasias de la Tiroides/genética , Regulación hacia Arriba , Adenocarcinoma Folicular/genética , Adenocarcinoma Folicular/patología , Animales , Carcinoma Papilar/patología , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Neoplasias de la Tiroides/patologíaRESUMEN
PURPOSE: Gastrointestinal stromal tumors (GIST) are characterized by gain-of-function mutations in KIT/PDGFRA genes leading to a constitutive receptor activation which is well counteracted by imatinib. However, cases in which imatinib as first-line treatment has no effects are reported (primary resistance). Our purpose is to investigate alterations in downstream effectors, not reported so far in mutated GIST, possibly explaining the primary resistance to targeted treatments. EXPERIMENTAL DESIGN: Two independent naive GIST cohorts have been analyzed for KIT, PDGFRA, KRAS, and BRAF mutations by direct sequencing. Cell lines expressing a constitutively activated and imatinib-responding KIT, alone or in combination with activated KRAS and BRAF, were produced and treated with imatinib. KIT receptor and its downstream effectors were analyzed by direct Western blotting. RESULTS: In naive GISTs carrying activating mutations in KIT or PDGFRA a concomitant activating mutation was detected in KRAS (5%) or BRAF (about 2%) genes. In vitro experiments showed that imatinib was able to switch off the mutated receptor KIT but not the downstream signaling triggered by RAS-RAF effectors. CONCLUSIONS: These data suggest the activation of mitogen-activated protein kinase pathway as a possible novel mechanism of primary resistance to imatinib in GISTs and could explain the survival curves obtained from several clinical studies where 2% to 4% of patients with GIST treated with imatinib, despite carrying KIT-sensitive mutations, do not respond to the treatment.
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Resistencia a Antineoplásicos/genética , Neoplasias Gastrointestinales/tratamiento farmacológico , Neoplasias Gastrointestinales/genética , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/genética , Genes ras , Mutación , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , Pirimidinas/uso terapéutico , Células 3T3 , Anciano , Animales , Benzamidas , Femenino , Células HEK293 , Humanos , Mesilato de Imatinib , Sistema de Señalización de MAP Quinasas/genética , Masculino , Ratones , Persona de Mediana EdadRESUMEN
Oncogene-induced senescence (OIS) is a growth arrest triggered by the enforced expression of cancer-promoting genes and acts as a barrier against malignant transformation in vivo. In this study, by a combination of in vitro and in vivo approaches, we investigate the role of OIS in tumours originating from the thyroid epithelium. We found that expression of different thyroid tumour-associated oncogenes in primary human thyrocytes triggers senescence, as demonstrated by the presence of OIS hallmarks: changes in cell morphology, accumulation of SA-ß-Gal and senescence-associated heterochromatic foci, and upregulation of transcription of the cyclin-dependent kinase inhibitors p16(INK4a) and p21(CIP1). Furthermore, immunohistochemical analysis of a panel of thyroid tumours characterised by different aggressiveness showed that the expression of OIS markers such as p16(INK4a), p21(CIP1) and IGFBP7 is upregulated at early stages, and lost during thyroid tumour progression. Taken together, our results suggest a role of OIS in thyroid carcinogenesis.
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Envejecimiento , Proto-Oncogenes , Neoplasias de la Tiroides/metabolismo , Adulto , Anciano , Carcinoma , Carcinoma Papilar , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Femenino , Células HEK293 , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Masculino , Persona de Mediana Edad , Proto-Oncogenes/genética , Cáncer Papilar Tiroideo , Carcinoma Anaplásico de Tiroides , Glándula Tiroides/citología , Transducción GenéticaRESUMEN
TRK oncoproteins are chimeric versions of the NTRK1/NGF receptor and display constitutive tyrosine kinase activity leading to transformation of NIH3T3 cells and neuronal differentiation of PC12 cells. Signal Transducer and Activator of Transcription (STAT) 3 is activated in response to cytokines and growth factors and it has been recently identified as a novel signal transducer for TrkA, mediating the functions of NGF in nervous system. In this paper we have investigated STAT3 involvement in signalling induced by TRK oncogenes. We showed that TRK oncogenes trigger STAT3 phosphorylation both on Y705 and S727 residues and STAT3 transcriptional activity. MAPK pathway was involved in the induction of STAT3 phosphorylation. Interestingly, we have shown reduced STAT3 protein level in NIH3T3 transformed foci expressing TRK oncogenes. Overall, we have unveiled a dual role for STAT3 in TRK oncogenes-induced NIH3T3 transformation: i) decreased STAT3 protein levels, driven by TRK oncoproteins activity, are associated to morphological transformation; ii) residual STAT3 transcriptional activity is required for cell growth.