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2.
J Vis Exp ; (190)2022 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-36622034

RESUMO

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.


Assuntos
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Camundongos , Animais , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço , Organoides/metabolismo , Linhagem Celular Tumoral , Proliferação de Células
3.
Acta Derm Venereol ; 100(10): adv00157, 2020 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-32449780

RESUMO

Although recent therapeutic developments raise hope, melanoma remains a devastating disease with a need for new treatment targets. In other tumours prohormone convertases have been shown to be pro-tumourigenic as they are involved in processing preforms of matrix-metalloproteinases, growth factors and adhesion molecules. The aim of this study was to look for new treatment options for melanoma, by investigating the role of the prohormone convertase Paired basic Amino acid-Cleaving Enzyme 4 (PACE4/PCSK6) in melanoma cell lines and human melanoma tissue. PACE4-transfected A375 melanoma cells displayed significantly increased proliferation, MMP-2 production, gelatinase activity and migratory capacity in vitro compared with sham-transfected cells. In vivo, elevated PACE4 expression resulted in significantly increased tumour growth on immunodeficient mice. In the majority of 45 human primary melanomas and melanoma metastases ex vivo PACE4 immunoreactivity was detectable, while it was absent in in situ melanomas. These results indicate PACE4 as a regulator of melanoma cell aggressiveness.


Assuntos
Melanoma/enzimologia , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Neoplasias Cutâneas/enzimologia , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Camundongos Pelados , Camundongos SCID , Terapia de Alvo Molecular , Invasividade Neoplásica , Pró-Proteína Convertases/antagonistas & inibidores , Pró-Proteína Convertases/genética , Serina Endopeptidases/genética , Inibidores de Serina Proteinase/uso terapêutico , Transdução de Sinais , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Carga Tumoral
5.
Biochem Pharmacol ; 164: 336-341, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30953636

RESUMO

Hypercholesterolemia represents a leading cause in the development of atherosclerotic plaques, increasing the risk for ACVS. It actually counts as a major cause of cardiovascular disease etiopathogenesis. The causes of hypercholesterolemia are multifactorial, spanning from genetic constitution, age, sex, to sedentary lifestyle and diets rich in sugars and lipids. Although dietary restriction in saturated fats, increased exercise, and other modification in lifestyle represent a first-line approach to treat very initial stages in hypercholesterolemia, most patients will require the addition of pharmacological agents. Pharmacological approaches include inhibition of cholesterol synthesis, decreased fat absorption from the GI tract, and increased degradation of FA. These strategies present a series of side effects, low therapeutic efficiency in some patients, and reduced tolerability. One of the major goals in treatment for hypercholesterolemia is to decrease the levels of low density lipoproteins (LDL), while maintaining those of high density lipoproteins (HDL). LDL particles contain about 80% of lipids, most of it cholesterol and cholesteryl esters, and 20% of the ApoB-100 protein. LDL carries cholesterol to the tissues, to be incorporated to biological membranes, or to be transformed to steroids. Excess of LDL translates into increased levels of circulating cholesterol particles and accumulation in certain tissues, especially vascular tissue, initiating a fatty streak, which may evolve to an atheroma, causing a series of cardiovascular problems, including impaired circulation, high blood pressure, increased cardiac workload, and coronary artery disease. It is essential to prevent LDL accumulation into the bloodstream to avoid the formation of these fatty streaks and the initiation of a cascade that will lead to the development of atherosclerosis. In healthy individuals. Under physiological conditions, LDL is effectively removed from circulation through receptor-mediated endocytosis. LDL clearance involves binding to its receptor, LDLR, which enables the internalization of the LDL particle and drives its degradation in lysosomes. Once the LDL particle is degraded, the free receptor recycles to the plasma membrane, and captures new LDL particles. Adequate levels of LDLR are essential to remove the excess of cholesterol-laden LDL. Proprotein convertase, subtilysin kexin type 9 (PCSK-9), expressed in liver and intestine, binds to LDLR, and internalized. Once inside the cell, PCSK-9 catalyzes the proteolysis of LDLR, preventing its recycling to the cell surface, and effectively decreasing the number of LDLR, notoriously decreasing the ability to clear LDL from circulation. Levels of PCSK-9 varies with age, gender, and levels of insulin, glucose, and triglycerides. Loss-of-function mutations in PCSK-9 gene invariably translates into lower levels of LDL, and decreased risk of developing coronary artery disease. Conversely, increased activity or expression of this enzyme leads to hypercholesterolemia. Inhibition of PCSK9 has proven to be successful in decreasing LDL levels and risk of the development of hypercholesterolemia with its associated higher risk for ASCVD. Patient with gain-of-function mutations in the PCSK9 undoubtedly benefit from therapies based on PCSK-9 inhibitors. However, millions of patients show statin intolerance, or cannot be efficiently controlled by statins alone- the most prevalent therapy for hypeprcholesterolemia. This commentary will evaluate the possibilities, caveats and future directions in the treatment of hypercholesterolemia, and therapies with combination of drugs.


Assuntos
Anticolesterolemiantes/uso terapêutico , LDL-Colesterol/antagonistas & inibidores , LDL-Colesterol/sangue , Hipercolesterolemia/sangue , Hipercolesterolemia/tratamento farmacológico , Animais , Anticolesterolemiantes/farmacologia , Aterosclerose/sangue , Aterosclerose/tratamento farmacológico , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/fisiologia
7.
Oncotarget ; 8(9): 14941-14956, 2017 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-28122332

RESUMO

Homeobox genes play a critical role in embryonic development, but they have also been implicated in cancer through mechanisms that are largely unknown. While not expressed during normal T-cell development, homeobox transcription factor genes can be reactivated via recurrent chromosomal rearrangements in human T-cell acute leukemia/lymphoma (T-ALL), a malignancy often associated with activated Notch and Akt signaling. To address how epigenetic reprogramming via an activated homeobox gene might contribute to T-lymphomagenesis, we investigated a transgenic mouse model with thymocyte-specific overexpression of the Dlx5 homeobox gene. We demonstrate for the first time that Dlx5 induces T-cell lymphomas with high penetrance. Integrated ChIP-seq and mRNA microarray analyses identified Notch1/3 and Irs2 as direct transcriptional targets of Dlx5, a gene signature unique to lymphomas from Lck-Dlx5 mice as compared to T-cell lymphomas from Lck-MyrAkt2 mice, which were previously reported by our group. Moreover, promoter/enhancer studies confirmed that Dlx5 directly transactivates Notch expression. Notch1/3 expression and Irs2-induced Akt signaling were upregulated throughout early stages of T-cell development, which promoted cell survival during ß-selection of T lymphocytes. Dlx5 was required for tumor maintenance via its activation of Notch and Akt, as tumor cells were highly sensitive to Notch and Akt inhibitors. Together, these findings provide unbiased genetic and mechanistic evidence that Dlx5 acts as an oncogene when aberrantly expressed in T cells, and that it is a novel discovery that Notch is a direct target of Dlx5. These experimental findings provide mechanistic insights about how reactivation of the Dlx5 gene can drive T-ALL by aberrant epigenetic reprogramming of the T-cell genome.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/fisiologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Linfoma de Células T/patologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptor Notch1/genética , Animais , Apoptose , Proliferação de Células , Humanos , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Transdução de Sinais , Ativação Transcricional , Células Tumorais Cultivadas
8.
Oncotarget ; 6(40): 42892-904, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26503472

RESUMO

The DNA glycosylase gene MBD4 safeguards genomic stability at CpG sites and is frequently mutated at coding poly-A tracks in mismatch repair (MMR)-defective colorectal tumors (CRC). Mbd4 biallelic inactivation in mice provided conflicting results as to its role in tumorigenesis. Thus, it is unclear whether MBD4 alterations are only secondary to MMR defects without functional consequences or can contribute to the mutator phenotype. We investigated MBD4 variants in a large series of hereditary/familial and sporadic CRC cases. Whereas MBD4 frameshifts were only detected in tumors, missense variants were found in both normal and tumor DNA. In CRC with double-MBD4/MMR and single-MBD4 variants, transition mutation frequency was increased, indicating that MBD4 defects may affect the mutational landscape independently of MMR defect. Mbd4-deficient mice showed reduced survival when combined with Mlh1-/- genotype. Taken together, these data suggest that MBD4 inactivation may contribute to tumorigenesis, acting as a modifier of MMR-deficient cancer phenotype.


Assuntos
Carcinogênese/genética , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Endodesoxirribonucleases/genética , Animais , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase
9.
Cancer Discov ; 5(3): 288-303, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25582697

RESUMO

UNLABELLED: Protein arginine methyltransferase 5 (PRMT5) has been implicated as a key modulator of lymphomagenesis. Whether PRMT5 has overt oncogenic function in the context of leukemia/lymphoma and whether it represents a therapeutic target remains to be established. We demonstrate that inactivation of PRMT5 inhibits colony-forming activity by multiple oncogenic drivers, including cyclin D1, c-MYC, NOTCH1, and MLL-AF9. Furthermore, we demonstrate that PRMT5 overexpression specifically cooperates with cyclin D1 to drive lymphomagenesis in a mouse model, revealing inherent neoplastic activity. Molecular analysis of lymphomas revealed that arginine methylation of p53 selectively suppresses expression of crucial proapoptotic and antiproliferative target genes, thereby sustaining tumor cell self-renewal and proliferation and bypassing the need for the acquisition of inactivating p53 mutations. Critically, analysis of human tumor specimens reveals a strong correlation between cyclin D1 overexpression and p53 methylation, supporting the biomedical relevance of this pathway. SIGNIFICANCE: We have identified and functionally validated a crucial role for PRMT5 for the inhibition of p53-dependent tumor suppression in response to oncogenic insults. The requisite role for PRMT5 in the context of multiple lymphoma/leukemia oncogenic drivers suggests a molecular rationale for therapeutic development.


Assuntos
Transformação Celular Neoplásica/genética , Linfoma/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Substituição de Aminoácidos , Animais , Apoptose/genética , Arginina/metabolismo , Transformação Celular Neoplásica/metabolismo , Análise por Conglomerados , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Ativação Enzimática , Perfilação da Expressão Gênica , Humanos , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , Linfoma/patologia , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Metilação , Camundongos , Mutação , Oncogenes , Fosforilação , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
10.
Head Neck ; 36(8): 1113-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23804308

RESUMO

BACKGROUND: Psychosocial functioning is associated with vascular endothelial growth factor (VEGF) in various patient populations. This study examined whether psychosocial functioning in patients with head and neck squamous cell carcinoma (HNSCC) is associated with tumor VEGF expression, a protein that stimulates angiogenesis and is associated with poor prognosis. METHODS: Forty-two newly diagnosed patients completed assessments of psychosocial functioning (ie, depressive symptoms, perceived stress, anxiety, social support) before surgery. Tumor samples were obtained for VEGF analysis and human papillomavirus (HPV)-typing. RESULTS: Poorer psychosocial functioning was associated with greater VEGF expression controlling for disease stage (odds ratio [OR], 4.55; 95% confidence interval [CI], 1.72-12.0; p < .01). When examined by HPV status, the association between psychosocial functioning and VEGF remained significant among patients who were HPV negative (OR, 5.50; 95% CI, 1.68-17.3; p < .01), but not among patients who were HPV positive. CONCLUSION: These findings inform our understanding of the biobehavioral pathways that may contribute to poor outcomes in non-HPV-associated HNSCCs.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Estresse Psicológico/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Transtornos de Ansiedade/metabolismo , Transtorno Depressivo/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço
11.
Cancer Res ; 72(22): 5966-75, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22983922

RESUMO

The RAS genes are the most commonly mutated oncogenes in human cancer and present a particular therapeutic dilemma, as direct targeting of Ras proteins by small molecules has proved difficult. Signaling pathways downstream of Ras, in particular Raf/Mek/Erk and PI3K/Akt/mTOR, are dominated by lipid and protein kinases that provide attractive alternate targets in Ras-driven tumors. As p21-activated kinase 1 (Pak1) has been shown to regulate both these signaling pathways and is itself upregulated in many human cancers, we assessed the role of Pak1 in Ras-driven skin cancer. In human squamous cell carcinoma (SCC), we found a strong positive correlation between advanced stage and grade and PAK1 expression. Using a mouse model of Kras-driven SCC, we showed that deletion of the mouse Pak1 gene led to markedly decreased tumorigenesis and progression, accompanied by near total loss of Erk and Akt activity. Treatment of Kras(G12D) mice with either of two distinct small molecule Pak inhibitors (PF3758309 and FRAX597) caused tumor regression and loss of Erk and Akt activity. Tumor regression was also seen in mice treated with a specific Mek inhibitor, but not with an Akt inhibitor. These findings establish Pak1 as a new target in KRAS-driven tumors and suggest a mechanism of action through the Erk, but not the Akt, signaling pathway.


Assuntos
Carcinoma de Células Escamosas/enzimologia , Proteínas Proto-Oncogênicas/metabolismo , Piridonas/farmacologia , Pirimidinas/farmacologia , Neoplasias Cutâneas/enzimologia , Quinases Ativadas por p21/biossíntese , Proteínas ras/metabolismo , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Deleção de Genes , Genes ras , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Gradação de Tumores , Estadiamento de Neoplasias , Proteína Oncogênica v-akt/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/deficiência , Quinases Ativadas por p21/genética , Proteínas ras/genética
12.
Neoplasia ; 14(4): 271-82, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22577343

RESUMO

Furin, one of the members of the family of proprotein convertases (PCs), ubiquitously expressed as a type I membrane-bound proteinase, activates several proteins that contribute to tumor progression. In vitro studies using cancer cell lines and clinical specimens demonstrated that furin processes important substrates such as insulin-like growth factor 1 receptor (IGF-1R) and transforming growth factor ß, leading to increased tumor growth and progression. Despite the numerous studies associating furin with tumor development, its effects in preclinical models has not been comprehensively studied. In this study, we sought to determine the protumorigenic role of furin in vivo after a two-stage chemical carcinogenesis protocol in transgenic mice in which furin expression was targeted to the epidermal basal layer. We found that processing of the PC substrate IGF-1R and the proliferation rate of mouse epidermis was enhanced in transgenic mice when compared with their WT counterparts. Histopathologic diagnoses of the tumors demonstrated that furin transgenic mice (line F47) developed twice as many squamous carcinomas as the control, WT mice (P < .002). Similarly, tumors cells from transgenic mice were able to process PC substrates more efficiently than tumor cells from WT mice. Furthermore, furin expression resulted in a higher SCC volume in transgenic mice as well as an increase in the percentage of high-grade SCC, including poorly differentiated and spindle cell carcinomas. In conclusion, expression of furin in the basal layer of the epidermis increased tumor development and enhanced tumor growth, supporting the consideration of furin as a potential target for cancer treatment.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Furina/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Feminino , Furina/genética , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Receptor IGF Tipo 1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia
13.
Mol Cell Biol ; 31(22): 4513-23, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21911473

RESUMO

Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complexes modulate the accumulation of key cell cycle regulatory proteins. Following the G(1)/S transition, SCF(Fbx4) targets cyclin D1 for proteasomal degradation, a critical event necessary for DNA replication fidelity. Deregulated cyclin D1 drives tumorigenesis, and inactivating mutations in Fbx4 have been identified in human cancer, suggesting that Fbx4 may function as a tumor suppressor. Fbx4(+/-) and Fbx4(-/-) mice succumb to multiple tumor phenotypes, including lymphomas, histiocytic sarcomas and, less frequently, mammary and hepatocellular carcinomas. Tumors and premalignant tissue from Fbx4(+/-) and Fbx4(-/-) mice exhibit elevated cyclin D1, an observation consistent with cyclin D1 as a target of Fbx4. Molecular dissection of the Fbx4 regulatory network in murine embryonic fibroblasts (MEFs) revealed that loss of Fbx4 results in cyclin D1 stabilization and nuclear accumulation throughout cell division. Increased proliferation in early passage primary MEFs is antagonized by DNA damage checkpoint activation, consistent with nuclear cyclin D1-driven genomic instability. Furthermore, Fbx4(-/-) MEFs exhibited increased susceptibility to Ras-dependent transformation in vitro, analogous to tumorigenesis observed in mice. Collectively, these data reveal a requisite role for the SCF(Fbx4) E3 ubiquitin ligase in regulating cyclin D1 accumulation, consistent with tumor suppressive function in vivo.


Assuntos
Transformação Celular Neoplásica , Ciclina D1/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Animais , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Ciclina D1/biossíntese , Ciclina D1/genética , Dano ao DNA , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
14.
Cell ; 146(1): 148-63, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21729786

RESUMO

Mechanotransduction is a key determinant of tissue homeostasis and tumor progression. It is driven by intercellular adhesions, cell contractility, and forces generated within the microenvironment and is dependent on extracellular matrix composition, organization, and compliance. We show that caveolin-1 (Cav1) favors cell elongation in three-dimensional cultures and promotes Rho- and force-dependent contraction, matrix alignment, and microenvironment stiffening through regulation of p190RhoGAP. In turn, microenvironment remodeling by Cav1 fibroblasts forces cell elongation. Cav1-deficient mice have disorganized stromal tissue architecture. Stroma associated with human carcinomas and melanoma metastases is enriched in Cav1-expressing carcinoma-associated fibroblasts (CAFs). Cav1 expression in breast CAFs correlates with low survival, and Cav1 depletion in CAFs decreases CAF contractility. Consistently, fibroblast expression of Cav1, through p190RhoGAP regulation, favors directional migration and invasiveness of carcinoma cells in vitro. In vivo, stromal Cav1 remodels peri- and intratumoral microenvironments to facilitate tumor invasion, correlating with increased metastatic potency. Thus, Cav1 modulates tissue responses through force-dependent architectural regulation of the microenvironment.


Assuntos
Caveolina 1/metabolismo , Metástase Neoplásica/patologia , Neoplasias/patologia , Animais , Movimento Celular , Fibroblastos/patologia , Humanos , Melanoma/patologia , Camundongos , Camundongos Knockout
15.
Cancer Res ; 71(6): 2250-9, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21406401

RESUMO

Antibody drugs are widely used in cancer therapy, but conditions to maximize tumor penetration and efficacy have yet to be fully elucidated. In this study, we investigated the impact of antibody binding affinity on tumor targeting and penetration with affinity variants that recognize the same epitope. Specifically, we compared four derivatives of the C6.5 monoclonal antibody (mAb), which recognizes the same HER2 epitope (monovalent K(D) values ranging from 270 to 0.56 nmol/L). Moderate affinity was associated with the highest tumor accumulation at 24 and 120 hours after intravenous injection, whereas high affinity was found to produce the lowest tumor accumulation. Highest affinity mAbs were confined to the perivascular space of tumors with an average penetration of 20.4 ± 7.5 µm from tumor blood vessels. Conversely, lowest affinity mAbs exhibited a broader distribution pattern with an average penetration of 84.8 ± 12.8 µm. In vitro internalization assays revealed that antibody internalization and catabolism generally increased with affinity, plateauing once the rate of HER2 internalization exceeded the rate of antibody dissociation. Effects of internalization and catabolism on tumor targeting were further examined using antibodies of moderate (C6.5) or high-affinity (trastuzumab), labeled with residualizing ((111)In-labeled) or nonresidualizing ((125)I-labeled) radioisotopes. Significant amounts of antibody of both affinities were degraded by tumors in vivo. Furthermore, moderate- to high-affinity mAbs targeting the same HER2 epitope with monovalent affinity above 23 nmol/L had equal tumor accumulation of residualizing radiolabel over 120 hours. Results indicated equal tumor exposure, suggesting that mAb penetration and retention in tumors reflected affinity-based differences in tumor catabolism. Together, these results suggest that high-density, rapidly internalizing antigens subject high-affinity antibodies to greater internalization and degradation, thereby limiting their penetration of tumors. In contrast, lower-affinity antibodies penetrate tumors more effectively when rates of antibody-antigen dissociation are higher than those of antigen internalization. Together, our findings offer insights into how to optimize the ability of therapeutic antibodies to penetrate tumors.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Neoplasias Ovarianas/imunologia , Receptor ErbB-2/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacocinética , Afinidade de Anticorpos/imunologia , Antígenos de Neoplasias/metabolismo , Ligação Competitiva , Linhagem Celular Tumoral , Endocitose/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Radioisótopos de Índio , Radioisótopos do Iodo , Camundongos , Camundongos SCID , Mutação , Transplante de Neoplasias , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Fatores de Tempo , Distribuição Tecidual , Transplante Heterólogo
16.
Cancer Prev Res (Phila) ; 4(1): 107-15, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21205741

RESUMO

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common type of cancer in the United States. The goal of this study was to evaluate the contribution of estrogens to the development of HNSCCs. Various cell lines derived from early- and late-stage head and neck lesions were used to characterize the expression of estrogen synthesis and metabolism genes, including cytochrome P450 (CYP) 1B1, examine the effect of estrogen on gene expression, and evaluate the role of CYP1B1 and/or estrogen in cell motility, proliferation, and apoptosis. Estrogen metabolism genes (CYP1B1, CYP1A1, catechol-o-methyltransferase, UDP-glucuronosyltransferase 1A1, and glutathione-S-transferase P1) and estrogen receptor (ER) ß were expressed in cell lines derived from both premalignant (MSK-Leuk1) and malignant (HNSCC) lesions. Exposure to estrogen induced CYP1B1 2.3- to 3.6-fold relative to vehicle-treated controls (P = 0.0004) in MSK-Leuk1 cells but not in HNSCC cells. CYP1B1 knockdown by shRNA reduced the migration and proliferation of MSK-Leuk1 cells by 57% and 45%, respectively. Exposure of MSK-Leuk1 cells to estrogen inhibited apoptosis by 26%, whereas supplementation with the antiestrogen fulvestrant restored estrogen-dependent apoptosis. Representation of the estrogen pathway in human head and neck tissues from 128 patients was examined using tissue microarrays. The majority of the samples exhibited immunohistochemical staining for ERß (91.9%), CYP1B1 (99.4%), and 17ß-estradiol (88.4%). CYP1B1 and ERß were elevated in HNSCCs relative to normal epithelium (P = 0.024 and 0.008, respectively). These data provide novel insight into the mechanisms underlying head and neck carcinogenesis and facilitate the identification of new targets for chemopreventive intervention.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Estrogênios/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Apoptose/efeitos dos fármacos , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Western Blotting , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1B1 , Estradiol/genética , Estradiol/metabolismo , Receptor beta de Estrogênio/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Técnicas Imunoenzimáticas , Leucoplasia/tratamento farmacológico , Leucoplasia/metabolismo , Leucoplasia/patologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de Tecidos , Células Tumorais Cultivadas
17.
Neoplasia ; 12(7): 516-26, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20651981

RESUMO

PACE4 is a proprotein convertase (PC) responsible for cleaving and activating proteins that contribute to enhance tumor progression. PACE4 overexpression significantly increased the susceptibility to carcinogenesis, leading to enhanced tumor cell proliferation and premature degradation of the basement membrane. In the present study, we sought to evaluate a novel approach to retard skin tumor progression based on the inhibition of PACE4. We used decanoyl-RVKR-chloromethylketone (CMK), a small-molecule PC inhibitor, for in vitro and in vivo experiments. We found that CMK-dependent blockage of PACE4 activity in skin squamous cell carcinoma cell lines resulted in impaired insulin-like growth factor 1 receptor maturation, diminished its intrinsic tyrosine kinase activity, and decreased tumor cell proliferation. Two-stage skin chemical carcinogenesis experiments, together with topical applications of CMK, demonstrated that this PC inhibitor markedly reduced tumor incidence, tumor multiplicity, and metastasis, pointing to a significant delay in tumor progression in wild-type and PACE4 transgenic mice. These results identify PACE4, together with other PCs, as suitable targets to slow down or block tumor progression, suggesting that PC inhibition is a potential approach for therapy for solid tumors.


Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Carcinoma de Células Escamosas/patologia , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Pró-Proteína Convertases/antagonistas & inibidores , Neoplasias Cutâneas/patologia , Pele/patologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular , Transformação Celular Neoplásica/patologia , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Pró-Proteína Convertases/genética , Pele/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
PLoS One ; 5(4): e10196, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20419170

RESUMO

VILIP-1, a member of the neuronal Ca(2+) sensor protein family, is able to act as a tumor suppressor in carcinoma cells by inhibiting cell proliferation and migration. In order to study the role of VILIP-1 in skin carcinogenesis we generated transgenic mice overexpressing VILIP-1 in epidermis under the control of the bovine keratin K5 promoter (K5-VILIP-1). We studied the susceptibility of FVB wild type and VILIP-1 transgenic mice to chemically mediated carcinogenesis. After 30 weeks of treatment with a two-stage carcinogenesis protocol, all animals showed numerous skin tumors. Nevertheless, K5-VILIP-1 mice showed decreased squamous cell carcinoma (SCC) multiplicity of approximately 49% (p<0.02) with respect to the corresponding SCC multiplicity observed in wild type (WT) mice. In addition, the relative percentage of low-grade cutaneous SCCs grade I (defined by the differentiation pattern according to the Broders grading scale) increased approximately 50% in the K5-VILIP1 mice when compared with SCCs in WT mice. Similar tendency was observed using a complete carcinogenesis protocol for skin carcinogenesis using benzo(a)pyrene (B(a)P). Further studies of tumors and primary epidermal keratinocyte cultures showed that matrix metalloproteinase 9 (MMP-9) levels and cell proliferation decreased in K5-VILIP-1 mice when compared with their wild counterparts. In addition tissue inhibitor of metalloproteinase 1 (TIMP-1) expression was higher in K5-VILIP-1 keratinocytes. These results show that VILIP-1 overexpression decreases the susceptibility to skin carcinogenesis in experimental mouse cancer models, thus supporting its role as a tumor suppressor gene.


Assuntos
Queratinócitos/citologia , Neurocalcina/fisiologia , Neoplasias Cutâneas/etiologia , Animais , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Suscetibilidade a Doenças , Genes Supressores de Tumor , Metaloproteinase 9 da Matriz , Camundongos , Camundongos Transgênicos , Neurocalcina/genética , Neoplasias Cutâneas/patologia , Inibidor Tecidual de Metaloproteinase-1 , Proteínas Supressoras de Tumor/genética
19.
Carcinogenesis ; 31(3): 427-34, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20042640

RESUMO

Hypoxia-inducible factors (HIFs), in particular HIF-1alpha, have been implicated in tumor biology. However, HIF target genes in the esophageal tumor microenvironment remain elusive. Gene expression profiling was performed upon hypoxia-exposed non-transformed immortalized human esophageal epithelial cells, EPC2-hTERT, and comparing with a gene signature of esophageal squamous cell carcinoma (ESCC). In addition to known HIF-1alpha target genes such as carbonic anhydrase 9, insulin-like growth factor binding protein-3 (IGFBP3) and cyclooxygenase (COX)-2, prostaglandin E synthase (PTGES) was identified as a novel target gene among the commonly upregulated genes in ESCC as well as the cells exposed to hypoxia. The PTGES induction was augmented upon stabilization of HIF-1alpha by hypoxia or cobalt chloride under normoxic conditions and suppressed by dominant-negative HIF-1alpha. Whereas PTGES messenger RNA (mRNA) was negatively regulated by normoxia, PTGES protein remained stable upon reoxygenation. Prostaglandin E(2) (PGE(2)) biosynthesis was documented in transformed human esophageal cells by ectopic expression of PTGES as well as RNA interference directed against PTGES. Moreover, hypoxia stimulated PGE(2) production in a HIF-1alpha-dependent manner. In ESCC, PTGES was overexpressed frequently at the mRNA and protein levels. Finally, COX-2 and PTGES were colocalized in primary tumors along with HIF-1alpha and IGFBP3. Activation of the COX-2-PTGES axis in primary tumors was further corroborated by concomitant upregulation of interleukin-1beta and downregulation of hydroxylprostaglandin dehydrogenase. Thus, PTGES is a novel HIF-1alpha target gene, involved in prostaglandin E biosynthesis in the esophageal tumor hypoxic microenvironment, and this has implications in diverse tumors types, especially of squamous origin.


Assuntos
Carcinoma de Células Escamosas/patologia , Hipóxia Celular/fisiologia , Neoplasias Esofágicas/patologia , Oxirredutases Intramoleculares/fisiologia , Proteínas de Neoplasias/fisiologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular , Cobalto/farmacologia , Ciclo-Oxigenase 2/fisiologia , Dinoprostona/biossíntese , Ativação Enzimática , Células Epiteliais/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Perfilação da Expressão Gênica , Humanos , Hidroxiprostaglandina Desidrogenases/biossíntese , Hidroxiprostaglandina Desidrogenases/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/fisiologia , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/biossíntese , Oxirredutases Intramoleculares/genética , Oxigênio/administração & dosagem , Oxigênio/metabolismo , Prostaglandina-E Sintases , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Células Tumorais Cultivadas/metabolismo
20.
J Clin Invest ; 118(1): 111-23, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18079962

RESUMO

Preclinical data support the potential of the death-signaling receptors for TRAIL as targets for cancer therapy. However, it is unclear whether these death-signaling receptors suppress the emergence and growth of malignant tumors in vivo. Herein we show that TNF-related apoptosis-inducing ligand receptor (TRAIL-R), the only proapoptotic death-signaling receptor for TRAIL in the mouse, suppresses inflammation and tumorigenesis. Loss of a single TRAIL-R allele on the lymphoma-prone Emu-myc genetic background significantly reduced median lymphoma-free survival. TRAIL-R-deficient lymphomas developed with equal frequency irrespective of mono- or biallelic loss of TRAIL-R, had increased metastatic potential, and showed apoptotic defects relative to WT littermates. In addition, TRAIL-R-/- mice showed decreased long-term survival following a sublethal dose of ionizing radiation. Histological evaluation of moribund irradiated TRAIL-R-/- animals showed hallmarks of bronchopneumonia as well as tumor formation with increased NF-kappaB p65 expression. TRAIL-R also suppressed diethylnitrosamine-induced (DEN-induced) hepatocarcinogenesis, as an increased number of large tumors with apoptotic defects developed in the livers of DEN-treated TRAIL-R-/- mice. Thus TRAIL-R may function as an inflammation and tumor suppressor in multiple tissues in vivo.


Assuntos
Bronquite/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias Hepáticas/metabolismo , Linfoma/metabolismo , Pneumonia/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Alquilantes/toxicidade , Animais , Bronquite/induzido quimicamente , Bronquite/genética , Bronquite/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Transformação Celular Neoplásica/efeitos da radiação , Doença Crônica , Dietilnitrosamina/toxicidade , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Linfoma/induzido quimicamente , Linfoma/genética , Linfoma/patologia , Camundongos , Camundongos Knockout , Pneumonia/induzido quimicamente , Pneumonia/genética , Pneumonia/patologia , Radiação Ionizante , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Proteínas Supressoras de Tumor/genética , Irradiação Corporal Total/efeitos adversos
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