RESUMO
Background: Several mechanisms likely cooperate with the mitogen-activated protein (MAP)-kinase pathway to promote cancer progression in the thyroid. One putative pathway is NOTCH signaling, which is implicated in several other malignancies. In thyroid cancer, data regarding the role of the NOTCH pathway are insufficient and even contradictory. Methods: A BRAFV600E-driven papillary thyroid carcinoma (PTC) mouse model was subjected to NOTCH pathway genetic alterations, and the tumor burden was followed by ultrasound. Further analyses were performed on PTC cell lines or noncancerous cells transfected with NOTCHIC or BRAFV600E, which were then subjected to pharmacological treatment with MAP-kinase or NOTCH pathway inhibitors. Results: The presence of the BRAFV600E mutation coupled with overexpression of the NOTCH intracellular domain led to significantly bigger thyroid tumors in mice, to a more aggressive carcinoma, and decreased overall survival. Although more cystic, the tumors did not progress into anaplastic thyroid carcinomas. On the contrary, the deletion of RBP-jκ (a major cofactor involved in NOTCH signaling) did not alter the phenotype in mice. BRAFV600E-mutated PTC cell lines were resistant to pharmacological inhibition of the NOTCH pathway. Inhibition of MEK1/2 uncovered a predominant effect on Hes1/Hey1 transcription compared with NOTCH inhibition in BRAFV600E-mutated cell lines. Finally, γ-secretase activity and γ-secretase subunit transcription levels were dependent on ERK activation. Our findings suggest that MAP-kinase activity overrides the NOTCH pathway in the context of thyroid cancer. Conclusions: The interaction between the BRAF and NOTCH pathways demonstrates that the BRAFV600E mutation might bypass NOTCH and exert a strong positive effect on NOTCH downstream targets in thyroid carcinoma.
Assuntos
Proteínas Proto-Oncogênicas B-raf/genética , Receptor Notch1/genética , Câncer Papilífero da Tireoide/genética , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Mutação , Receptor Notch1/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Câncer Papilífero da Tireoide/patologia , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Fatores de Transcrição HES-1/metabolismo , Carga TumoralRESUMO
The solute carrier 16 (SLC16) family represents a diverse group of membrane proteins mediating the transport of monocarboxylates across biological membranes. Family members show a variety of functional roles ranging from nutrient transport and intracellular pH regulation to thyroid hormone homeostasis. Changes in the expression levels and transport function of certain SLC16 transporters are manifested in severe health disorders including cancer, diabetes, and neurological disorders. L-Lactate-transporting SLC16 family members play essential roles in the metabolism of certain tumors and became validated drug targets. This review illuminates the SLC16 family under a new light using structural information obtained from a SLC16 homolog. Furthermore, the role of these transporters in cancer metabolism and how their inhibition can contribute to anticancer therapy are discussed.
Assuntos
Transportadores de Ácidos Monocarboxílicos/química , Simportadores/química , Transporte Biológico , Humanos , Transportadores de Ácidos Monocarboxílicos/genética , Conformação Proteica , Simportadores/genética , Difração de Raios XRESUMO
Chronic proliferation is a major hallmark of tumor cells. Rapidly proliferating cancer cells are highly dependent on nutrients in order to duplicate their cell mass during each cell division. In particular, essential amino acids are indispensable for proliferating cancer cells. Their uptake across the cell membrane is tightly controlled by membrane transporters. Among those, the L-type amino acid transporter LAT1 (SLC7A5) has been repeatedly found overexpressed in a vast variety of cancers. In this review, we summarize the most recent advances in our understanding of the role of LAT1 in cancer and highlight preclinical studies and drug developments underlying the potential of LAT1 as therapeutic target.
Assuntos
Aminoácidos/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Neoplasias/metabolismo , Sistema y+L de Transporte de Aminoácidos , Transporte Biológico , Proliferação de Células , Descoberta de Drogas , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/efeitos dos fármacos , Nanopartículas/química , Proteínas de NeoplasiasRESUMO
Background: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers, with a median survival of only three to six months. Standard treatment options and even targeted therapies have so far failed to improve long-term overall survival. Thus, novel treatment modalities for ATC, such as immunotherapy, are urgently needed. CD47 is a "don't eat me" signal, which prevents cancer cells from phagocytosis by binding to signal regulatory protein alpha on macrophages. So far, the role of macrophages and the CD47-signal regulatory protein alpha signaling axis in ATC is not well understood. Methods: This study analyzed 19 primary human ATCs for macrophage markers, CD47 expression, and immune checkpoints by immunohistochemistry. ATC cell lines and a fresh ATC sample were assessed by flow cytometry for CD47 expression and macrophage infiltration, respectively. CD47 was blocked in phagocytosis assays of co-cultured macrophages and ATC cell lines. Anti-CD47 antibody treatment was administered to ATC cell line xenotransplanted immunocompromised mice, as well as to tamoxifen-induced ATC double-transgenic mice. Results: Human ATC samples were heavily infiltrated by CD68- and CD163-expressing tumor-associated macrophages (TAMs), and expressed CD47 and calreticulin, the dominant pro-phagocytic molecule. In addition, ATC tissues expressed the immune checkpoint molecules programmed cell death 1 and programmed death ligand 1. Blocking CD47 promoted the phagocytosis of ATC cell lines by macrophages in vitro. Anti-CD47 antibody treatment of ATC xenotransplanted mice increased the frequency of TAMs, enhanced the expression of macrophage activation markers, augmented tumor cell phagocytosis, and suppressed tumor growth. In double-transgenic ATC mice, CD47 was expressed on tumor cells, and blocking CD47 increased TAM frequencies. Conclusions: Targeting CD47 or CD47 in combination with programmed cell death 1 may potentially improve the outcomes of ATC patients and may represent a valuable addition to the current standard of care.
Assuntos
Antígenos de Diferenciação/imunologia , Antígeno CD47/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Receptores Imunológicos/imunologia , Carcinoma Anaplásico da Tireoide/imunologia , Neoplasias da Glândula Tireoide/imunologia , Evasão Tumoral/imunologia , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos de Diferenciação/metabolismo , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Imunoterapia , Técnicas In Vitro , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Transplante de Neoplasias , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores Imunológicos/metabolismo , Carcinoma Anaplásico da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anaplastic thyroid carcinoma (ATC) is refractory to radioiodine therapy in part because of impaired iodine metabolism. We targeted the mitogen-activated protein kinase and phosphatidylinositol 3-kinase (PI3'K) pathways with the intent to induce radioiodine uptake for radioiodine treatment of ATC. Methods: Human ATC cells were used to evaluate the ability of pharmacologic inhibition of the mitogen-activated protein kinase and PI3'K pathways to induce radioiodine uptake. Thyrocyte-specific double-mutant BRAFV600E PIK3CAH1047R mice were treated with a MEK inhibitor followed by radioiodine treatment, and tumor burden was monitored by ultrasound imaging. Results: ATC cell lines showed an increase in sodium-iodine symporter transcription when treated with a MEK or BRAFV600E inhibitor alone and in combination with PI3'K inhibitor. This translated into a dose-dependent elevation of iodine uptake after treatment with a MEK inhibitor alone and in combination with a PI3'K inhibitor. In vivo, MEK inhibition but not BRAF or PI3'K inhibition upregulated sodium-iodine symporter transcription. This translated into a stable reduction of tumor burden when mice were treated with a MEK inhibitor before radioiodine administration. Conclusion: This study confirms the ability of MEK inhibition to induce iodine uptake in in vitro and in vivo models of ATC. The approach of using a MEK inhibitor before radioiodine treatment could readily be translated into clinical practice and provide a much-needed therapeutic option for patients with ATC.
Assuntos
Radioisótopos do Iodo/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Carcinoma Anaplásico da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Radioisótopos do Iodo/uso terapêutico , Camundongos , RNA Mensageiro/genética , Simportadores/genética , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/patologia , Carcinoma Anaplásico da Tireoide/radioterapia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/radioterapia , Transcrição Gênica/efeitos dos fármacosRESUMO
By screening a focused library of kinase inhibitor analogues in a phenotypic co-culture assay for angiogenesis inhibition, we identified an aminotriazine that acts as a cytostatic nanomolar inhibitor. However, this aminotriazine was found to be completely inactive in a whole-kinome profiling assay. To decipher its mechanism of action, we used the online target prediction tool PPB2 (http://ppb2.gdb.tools), which suggested lysophosphatidic acid acyltransferaseâ ß (LPAAT-ß) as a possible target for this aminotriazine as well as several analogues identified by structure-activity relationship profiling. LPAAT-ß inhibition (IC50 ≈15â nm) was confirmed in a biochemical assay and by its effects on cell proliferation in comparison with a known LPAAT-ß inhibitor. These experiments illustrate the value of target-prediction tools to guide target identification for phenotypic screening hits and significantly expand the rather limited pharmacology of LPAAT-ß inhibitors.
Assuntos
Aciltransferases/antagonistas & inibidores , Indutores da Angiogênese/metabolismo , Inibidores Enzimáticos/química , Bibliotecas de Moléculas Pequenas/química , Triazinas/química , Aciltransferases/genética , Aciltransferases/isolamento & purificação , Bioensaio/métodos , Técnicas de Cultura de Células , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Fenótipo , Ligação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo , Software , Relação Estrutura-Atividade , Triazinas/metabolismoRESUMO
BACKGROUND: The L-type amino acid transporter 1 (LAT1/SLC7A5) transports essential amino acids across the plasma membrane. While LAT1 is overexpressed in a variety of human neoplasms, its expression and its role in thyroid cancer is currently unknown. Anaplastic thyroid carcinoma (ATC) is a highly aggressive malignancy for which no effective therapy exists. The purpose of this study was to explore whether the inhibition of LAT1 in ATC would affect tumor growth both in vitro and in vivo. METHODS: LAT1 was pharmacologically blocked by JPH203 in human ATC and papillary thyroid cancer (PTC) cell lines. The effects on proliferation and mTORC1 activity were addressed in vitro. A genetically engineered mouse model of ATC was used to address the effect of blocking LAT1 on tumor growth in vivo. SLC7A5 transcription was measured in patient-derived ATC samples to address the clinical relevance of the findings. RESULTS: LAT1 block by JPH203 reduced proliferation and mTORC1 signaling in human thyroid cancer cell lines. SLC7A5 transcription was upregulated in ATC tissues derived from a genetically engineered mouse model and in ATC samples recovered from patients. JPH203 treatment induced thyroid tumor growth arrest in vivo in a fully immunocompetent mouse model of thyroid cancer. Additionally, analysis of publicly available datasets of thyroid carcinomas revealed that high LAT1 expression is associated with potentially untreatable PTC presenting reduced NIS/SLC5A5 transcription and with ATC. CONCLUSIONS: These preclinical results show that LAT1 inhibition is a novel therapeutic approach in the context of thyroid cancers, and more interestingly in untreatable thyroid cancers.
Assuntos
Proliferação de Células/efeitos dos fármacos , Transportador 1 de Aminoácidos Neutros Grandes/genética , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Animais , Animais Geneticamente Modificados/genética , Apoptose/efeitos dos fármacos , Benzoxazóis/administração & dosagem , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/efeitos dos fármacos , Camundongos , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais/efeitos dos fármacos , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/patologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/patologia , Tirosina/administração & dosagem , Tirosina/análogos & derivadosRESUMO
Thyroid carcinomas are the most prevalent endocrine cancers. The BRAFV600E mutation is found in 40% of the papillary type and 25% of the anaplastic type. BRAFV600E inhibitors have shown great success in melanoma but, they have been, to date, less successful in thyroid cancer. About 50% of anaplastic thyroid carcinomas present mutations/amplification of the phosphatidylinositol 3' kinase. Here we propose to investigate if the hyper activation of that pathway could influence the response to BRAFV600E specific inhibitors. To test this, we used two mouse models of thyroid cancer. Single mutant (BRAFV600E) mice responded to BRAFV600E-specific inhibition (PLX-4720), while double mutant mice (BRAFV600E; PIK3CAH1047R) showed resistance and even signs of aggravation. This resistance was abrogated by combination with a phosphoinositide 3-kinase inhibitor. At the molecular level, we showed that this resistance was concomitant to a paradoxical activation of the MAP-Kinase pathway, which could be overturned by phosphoinositide 3-kinase inhibition in vivo in our mouse model and in vitro in human double mutant cell lines. In conclusion, we reveal a phosphoinositide 3-kinase driven, paradoxical MAP-Kinase pathway activation as mechanism for resistance to BRAFV600E specific inhibitors in a clinically relevant mouse model of thyroid cancer.
RESUMO
PI3K signaling is frequently dysregulated in NSCLC-SQCC. In contrast to well characterized components of the PI3K signaling network contributing to the formation of SQCC, potential oncogenic effects of alterations in PIK3C2B are poorly understood. Here, a large cohort (n = 362) of NSCLC-SQCC was selectively screened for four reported somatic mutations in PIK3C2B via Sanger sequencing. In addition, two mutations leading to an amino acid exchange in the kinase domain (C1181, H1208R) were examined on a functional level. None of the mutations were identified in the cohort while well characterized hotspot PIK3CA mutations were observed at the expected frequency. Ultimately, kinase domain mutations in PI3KC2ß were found to have no altering effect on downstream signaling. A set of SQCC tumors sequenced by The Cancer Genome Atlas (TCGA) equally indicates a lack of oncogenic potential of the kinase domain mutations or PIK3C2B in general. Taken together, this study suggests that PIK3C2B might only have a minor role in SQCC oncogenesis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Classe II de Fosfatidilinositol 3-Quinases/genética , Neoplasias Pulmonares/genética , Mutação , Carcinogênese , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos de Coortes , Células HEK293 , Humanos , Neoplasias Pulmonares/patologiaRESUMO
The extracellular effects of the endocannabinoids anandamide and 2-arachidonoyl glycerol are terminated by enzymatic hydrolysis after crossing cellular membranes by facilitated diffusion. The lack of potent and selective inhibitors for endocannabinoid transport has prevented the molecular characterization of this process, thus hindering its biochemical investigation and pharmacological exploitation. Here, we report the design, chemical synthesis, and biological profiling of natural product-derived N-substituted 2,4-dodecadienamides as a selective endocannabinoid uptake inhibitor. The highly potent (IC50 = 10 nM) inhibitor N-(3,4-dimethoxyphenyl)ethyl amide (WOBE437) exerted pronounced cannabinoid receptor-dependent anxiolytic, antiinflammatory, and analgesic effects in mice by increasing endocannabinoid levels. A tailored WOBE437-derived diazirine-containing photoaffinity probe (RX-055) irreversibly blocked membrane transport of both endocannabinoids, providing mechanistic insights into this complex process. Moreover, RX-055 exerted site-specific anxiolytic effects on in situ photoactivation in the brain. This study describes suitable inhibitors to target endocannabinoid membrane trafficking and uncovers an alternative endocannabinoid pharmacology.
Assuntos
Transporte Biológico/efeitos dos fármacos , Endocanabinoides/metabolismo , Animais , Ansiolíticos/farmacologia , Anti-Inflamatórios/farmacologia , Ácidos Araquidônicos/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Glicerídeos/metabolismo , Humanos , Hidrólise/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Alcamidas Poli-Insaturadas/metabolismo , Receptores de Canabinoides/metabolismo , Células U937RESUMO
BACKGROUND: The MET receptor tyrosine kinase represents a promising target in cancer. PIK3CA activating mutations are common in several tumor types and can potentially confer resistance to anti-receptor tyrosine kinase therapy. METHODS: MET and/or PI3K pathway inhibition was assessed in NIH3T3 cells harboring MET-activating point mutation with or without ectopic expression of PIK3CAE545K and PIK3CAH1047R, as well as in MET-expressing head and neck cancer cells with endogenous PIK3CA mutations. Endpoints included PI3K pathway activation, cell proliferation, colony-forming ability, cell death, wound-healing, and an in vivo model. RESULTS: PIK3CAE545K and PIK3CAH1047R confer resistance to MET inhibition in MET-driven models. PIK3CAH1047R was more potent than PIK3CAE545K at inducing resistance in PI3K pathway activation, cell proliferation, colony-forming ability, induction of cell death and wound-healing upon MET inhibition. Resistance to MET inhibition could be synergistically overcome by co-targeting PI3K. Furthermore, combined MET/PI3K inhibition led to enhanced anti-tumor activity in vivo in tumors harboring PIK3CAH1047R. In head and neck cancer cells the combination of MET/PI3K inhibitors led to more-than-additive effects. CONCLUSIONS: PIK3CA mutations can lead to resistance to MET inhibition, supporting future clinical evaluation of combinations of PI3K and MET inhibitors in common scenarios of malignant neoplasms featuring aberrant MET expression and PIK3CA mutations.
Assuntos
Mutação , Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Camundongos , Células NIH 3T3 , Neoplasias/metabolismo , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anaplastic thyroid cancers and radioiodine resistant thyroid cancer are posing a major treat since surgery combined with Iodine131 therapy is ineffective on them. Small-molecule inhibitors are presenting a new hope for patients, but often lead to drug resistance in many cancers. Based on the major mutations found in thyroid cancer, we propose the combination of a MEK inhibitor and a Pi3'-kinase inhibitor in pre-clinical models. We used human thyroid cancer cell lines and genetically engineered double mutant BRAFV600E PIK3CAH1047R mice to evaluate the effect of both inhibitors separately or in combination in terms of proliferation and signaling in vitro; tumor burden, histology, cell death induction and tumor markers expression in vivo. The combination of MEK and Pi'3-kinase inhibition shows a synergistic effect in term of proliferation and apoptosis induction through Survivin down-regulation in vitro. We show for the first time the effects of the combination of a MEK inhibitor and Pi3'-kinase inhibitor in a genetically engineered mouse model of aggressively lethal thyroid cancer. In fine, the two drugs cooperate to promote tumor shrinkage by inducing a proliferation arrest and an elevation of apoptosis in vivo. Moreover, a phenotypic reversion is also observed with a partial restoration of normal thyroid marker transcription, and thyroid cancer marker expression reduction.In conclusion, combination therapy of MEK and Pi3'-kinase inhibition synergizes to target double mutant thyroid cancer in vitro and in vivo. This multidrug approach could readily be translated into clinical practice and bring new perspectives for the treatment of incurable thyroid carcinoma.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Neoplasias da Glândula Tireoide/genéticaRESUMO
Herein, we report the discovery of the first potent and selective inhibitor of TRPV6, a calcium channel overexpressed in breast and prostate cancer, and its use to test the effect of blocking TRPV6-mediated Ca(2+)-influx on cell growth. The inhibitor was discovered through a computational method, xLOS, a 3D-shape and pharmacophore similarity algorithm, a type of ligand-based virtual screening (LBVS) method described briefly here. Starting with a single weakly active seed molecule, two successive rounds of LBVS followed by optimization by chemical synthesis led to a selective molecule with 0.3â µM inhibition of TRPV6. The ability of xLOS to identify different scaffolds early in LBVS was essential to success. The xLOS method may be generally useful to develop tool compounds for poorly characterized targets.
Assuntos
Antineoplásicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Canais de Cátion TRPV/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Bloqueadores dos Canais de Cálcio/síntese química , Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio/biossíntese , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Estrutura Molecular , Relação Estrutura-Atividade , Canais de Cátion TRPV/biossínteseRESUMO
The prognosis from thyroid cancer subtypes in humans covers a spectrum from "cured at almost 90%" to "100% lethal." Invasive and poorly differentiated forms of thyroid cancer are among the most aggressive human cancers, and there are few effective therapeutic options. Genetically engineered mice, based on mutations observed in patients, can accurately recapitulate the human disease and its progression, providing invaluable tools for the preclinical evaluation of novel therapeutic approaches. This overview details models developed to date as well as their uses for identifying novel anticancer agents.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Papilar/tratamento farmacológico , Modelos Animais de Doenças , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Pesquisa Translacional Biomédica , Animais , Antineoplásicos/farmacologia , Carcinoma/tratamento farmacológico , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Humanos , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Câncer Papilífero da Tireoide , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Pesquisa Translacional Biomédica/tendências , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND PURPOSE: 4'-O-methylhonokiol (MH) is a natural product showing anti-inflammatory, anti-osteoclastogenic, and neuroprotective effects. MH was reported to modulate cannabinoid CB2 receptors as an inverse agonist for cAMP production and an agonist for intracellular [Ca2+]. It was recently shown that MH inhibits cAMP formation via CB2 receptors. In this study, the exact modulation of MH on CB2 receptor activity was elucidated and its endocannabinoid substrate-specific inhibition (SSI) of cyclooxygenase-2 (COX-2) and CNS bioavailability are described for the first time. METHODS: CB2 receptor modulation ([35S]GTPγS, cAMP, and ß-arrestin) by MH was measured in hCB2-transfected CHO-K1 cells and native conditions (HL60 cells and mouse spleen). The COX-2 SSI was investigated in RAW264.7 cells and in Swiss albino mice by targeted metabolomics using LC-MS/MS. RESULTS: MH is a CB2 receptor agonist and a potent COX-2 SSI. It induced partial agonism in both the [35S]GTPγS binding and ß-arrestin recruitment assays while being a full agonist in the cAMP pathway. MH selectively inhibited PGE2 glycerol ester formation (over PGE2) in RAW264.7 cells and significantly increased the levels of 2-AG in mouse brain in a dose-dependent manner (3 to 20 mg kg(-1)) without affecting other metabolites. After 7 h from intraperitoneal (i.p.) injection, MH was quantified in significant amounts in the brain (corresponding to 200 to 300 nM). CONCLUSIONS: LC-MS/MS quantification shows that MH is bioavailable to the brain and under condition of inflammation exerts significant indirect effects on 2-AG levels. The biphenyl scaffold might serve as valuable source of dual CB2 receptor modulators and COX-2 SSIs as demonstrated by additional MH analogs that show similar effects. The combination of CB2 agonism and COX-2 SSI offers a yet unexplored polypharmacology with expected synergistic effects in neuroinflammatory diseases, thus providing a rationale for the diverse neuroprotective effects reported for MH in animal models.
Assuntos
Anti-Inflamatórios/farmacologia , Ácidos Araquidônicos/metabolismo , Compostos de Bifenilo/farmacologia , Encéfalo/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Lignanas/farmacologia , Animais , Ácidos Araquidônicos/farmacocinética , Arrestinas/metabolismo , Encéfalo/metabolismo , Células CHO , Linhagem Celular Transformada , Cricetulus , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Endocanabinoides/farmacocinética , Feminino , Glicerídeos/farmacocinética , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Macrófagos , Camundongos , Elastômeros de Silicone/farmacocinética , Isótopos de Enxofre/farmacocinética , Trítio/farmacocinética , beta-ArrestinasRESUMO
UNLABELLED: Thyroid malignancies are the most common type of endocrine tumors. Of the various histologic subtypes, anaplastic thyroid carcinoma (ATC) represents a subset of all cases but is responsible for a significant proportion of thyroid cancer-related mortality. Indeed, ATC is regarded as one of the more aggressive and hard to treat forms of cancer. To date, there is a paucity of relevant model systems to critically evaluate how the signature genetic abnormalities detected in human ATC contribute to disease pathogenesis. Mutational activation of the BRAF protooncogene is detected in approximately 40% of papillary thyroid carcinoma (PTC) and in 25% of ATC. Moreover, in ATC, mutated BRAF is frequently found in combination with gain-of-function mutations in the p110 catalytic subunit of PI3'-Kinase (PIK3CA) or loss-of-function alterations in either the p53 (TP53) or PTEN tumor suppressors. Using mice with conditional, thyrocyte-specific expression of BRAF(V600E), we previously developed a model of PTC. However, as in humans, BRAF(V600E)-induced mouse PTC is indolent and does not lead to rapid development of end-stage disease. Here, we use mice carrying a conditional allele of PIK3CA to demonstrate that, although mutationally activated PIK3CA(H1047R) is unable to drive transformation on its own, when combined with BRAF(V600E) in thyrocytes, this leads to development of lethal ATC in mice. Combined, these data demonstrate that the BRAF(V600E) cooperates with either PIK3CA(H1074R) or with silencing of the tumor-suppressor PTEN, to promote development of anaplastic thyroid carcinoma. IMPLICATIONS: This genetically relevant mouse model of ATC will be an invaluable platform for preclinical testing of pathway-targeted therapies for the prevention and treatment of thyroid carcinoma.
Assuntos
Mutação , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Carcinoma Anaplásico da Tireoide/genética , Animais , Carcinogênese/genética , Processos de Crescimento Celular/genética , Classe I de Fosfatidilinositol 3-Quinases , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Análise de Sobrevida , Carcinoma Anaplásico da Tireoide/enzimologia , Carcinoma Anaplásico da Tireoide/patologiaRESUMO
Aldosterone promotes electrogenic sodium reabsorption through the amiloride-sensitive epithelial sodium channel (ENaC). Here, we investigated the importance of ENaC and its positive regulator channel-activating protease 1 (CAP1/Prss8) in colon. Mice lacking the αENaC subunit in colonic superficial cells (Scnn1a(KO)) were viable, without fetal or perinatal lethality. Control mice fed a regular or low-salt diet had a significantly higher amiloride-sensitive rectal potential difference (∆PDamil) than control mice fed a high-salt diet. In Scnn1a(KO) mice, however, this salt restriction-induced increase in ∆PDamil did not occur, and the circadian rhythm of ∆PDamil was blunted. Plasma and urinary sodium and potassium did not change with regular or high-salt diets or potassium loading in control or Scnn1a(KO) mice. However, Scnn1a(KO) mice fed a low-salt diet lost significant amounts of sodium in their feces and exhibited high plasma aldosterone and increased urinary sodium retention. Mice lacking the CAP1/Prss8 in colonic superficial cells (Prss8(KO)) were viable, without fetal or perinatal lethality. Compared with controls, Prss8(KO) mice fed regular or low-salt diets exhibited significantly reduced ∆PDamil in the afternoon, but the circadian rhythm was maintained. Prss8(KO) mice fed a low-salt diet also exhibited sodium loss through feces and higher plasma aldosterone levels. Thus, we identified CAP1/Prss8 as an in vivo regulator of ENaC in colon. We conclude that, under salt restriction, activation of the renin-angiotensin-aldosterone system in the kidney compensated for the absence of ENaC in colonic surface epithelium, leading to colon-specific pseudohypoaldosteronism type 1 with mineralocorticoid resistance without evidence of impaired potassium balance.
Assuntos
Aldosterona/metabolismo , Colo/metabolismo , Canais Epiteliais de Sódio/fisiologia , Sódio/metabolismo , Animais , Canais Epiteliais de Sódio/deficiência , Feminino , Masculino , Camundongos , Serina Endopeptidases/fisiologiaRESUMO
Cellular directional migration in an electric field (galvanotaxis) is one of the mechanisms guiding cell movement in embryogenesis and in skin epidermal repair. The epithelial sodium channel (ENaC), in addition to its function of regulating sodium transport in kidney, has recently been found to modulate cell locomotory speed. Here we tested whether ENaC has an additional function of mediating the directional migration of galvanotaxis in keratinocytes. Genetic depletion of ENaC completely blocks only galvanotaxis and does not decrease migration speed. Overexpression of ENaC is sufficient to drive galvanotaxis in otherwise unresponsive cells. Pharmacologic blockade or maintenance of the open state of ENaC also decreases or increases, respectively, galvanotaxis, suggesting that the channel open state is responsible for the response. Stable lamellipodial extensions formed at the cathodal sides of wild-type cells at the start of galvanotaxis; these were absent in the ENaC knockout keratinocytes, suggesting that ENaC mediates galvanotaxis by generating stable lamellipodia that steer cell migration. We provide evidence that ENaC is required for directional migration of keratinocytes in an electric field, supporting a role for ENaC in skin wound healing.
Assuntos
Movimento Celular , Canais Epiteliais de Sódio/metabolismo , Queratinócitos/metabolismo , Sódio/metabolismo , Animais , Canais Epiteliais de Sódio/genética , Técnicas de Silenciamento de Genes , Humanos , Transporte de Íons/genética , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Knockout , Pele/lesões , Pele/metabolismo , Pele/patologia , Cicatrização/genéticaRESUMO
The serine protease CAP1/Prss8 is crucial for skin barrier function, lung alveolar fluid clearance and has been unveiled as diagnostic marker for specific cancer types. Here, we show that a constitutive knockout of CAP1/Prss8 leads to embryonic lethality. These embryos presented no specific defects, but it is during this period, and in particular at E13.5, that wildtype placentas show an increased expression of CAP1/Prss8, thus suggesting a placental defect in the knockout situation. The placentas of knockout embryos exhibited significantly reduced vascular development and incomplete cellular maturation. In contrary, epiblast-specific deletion of CAP1/Prss8 allowed development until birth. These CAP1/Prss8-deficient newborns presented abnormal epidermis, and died soon after birth due to impaired skin function. We thus conclude that a late placental insufficiency might be the primary cause of embryonic lethality in CAP1/Prss8 knockouts. This study highlights a novel and crucial role for CAP1/Prss8 in placental development and function.