RESUMO
The AKT kinases are critical signaling molecules that regulate cellular physiology upon the activation of tyrosine kinase receptors and phosphatidylinositol 3-kinases (PI3K). AKT kinases govern many cellular processes considered hallmarks of cancer, including cell proliferation and survival, cell size, tumor invasion, metastasis, and angiogenesis. AKT signaling is regulated by multiple tumor suppressors and oncogenic proteins whose loss or activation, respectively, leads to dysregulation of this pathway, thereby contributing to oncogenesis. Herein, we review the enormous body of literature documenting how the AKT pathway becomes hyperactivated in sporadic human tumors and various hereditary cancer syndromes. We also discuss the role of activating mutations of AKT pathway genes in various chimeric overgrowth disorders, including Proteus syndrome, hypoglycemia with hypertrophy, CLOVES and SOLAMEN syndromes, and hemimegalencephaly.
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Because loss of the NF2 tumor suppressor gene results in p21-activated kinase (Pak) activation, PAK inhibitors hold promise for the treatment of NF2-deficient tumors. To test this possibility, we asked if loss of Pak2, a highly expressed group I PAK member, affects the development of malignant mesothelioma in Nf2;Cdkn2a-deficient (NC) mice and the growth properties of NC mesothelioma cells in culture. In vivo, deletion of Pak2 resulted in a markedly decreased incidence and delayed onset of both pleural and peritoneal malignant mesotheliomas in NC mice. In vitro, Pak2 deletion decreased malignant mesothelioma cell viability, migration, clonogenicity, and spheroid formation. RNA-sequencing analysis demonstrated downregulated expression of Hedgehog and Wnt pathway genes in NC;Pak2-/- mesothelioma cells versus NC;Pak2+/+ mesothelioma cells. Targeting of the Hedgehog signaling component Gli1 or its target gene Myc inhibited cell viability and spheroid formation in NC;P+/+ mesothelioma cells. Kinome profiling uncovered kinase changes indicative of EMT in NC;Pak2-/- mesothelioma cells, suggesting that Pak2-deficient malignant mesotheliomas can adapt by reprogramming their kinome in the absence of Pak activity. The identification of such compensatory pathways offers opportunities for rational combination therapies to circumvent resistance to anti-PAK drugs. IMPLICATIONS: We provide evidence supporting a role for PAK inhibitors in treating NF2-deficient tumors. NF2-deficient tumors lacking Pak2 eventually adapt by kinome reprogramming, presenting opportunities for combination therapies to bypass anti-PAK drug resistance.
Assuntos
Mesotelioma Maligno , Mesotelioma , Animais , Proteínas Hedgehog/genética , Humanos , Mesotelioma/tratamento farmacológico , Mesotelioma/genética , Camundongos , Via de Sinalização Wnt , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismoRESUMO
PURPOSE: Receptor-interacting protein kinase 3 (RIPK3) phosphorylates effector molecule MLKL to trigger necroptosis. Although RIPK3 loss is seen in several human cancers, its role in malignant mesothelioma is unknown. This study aimed to determine whether RIPK3 functions as a potential tumor suppressor to limit development of malignant mesothelioma. EXPERIMENTAL DESIGN: RIPK3 expression was examined in 66 malignant mesothelioma tumors and cell lines. Promoter methylation and DNMT1 siRNA studies were performed to assess the mode of RIPK3 silencing in RIPK3-deficient malignant mesothelioma cells. Restoration of RIPK3 expression in RIPK3-negative malignant mesothelioma cells, either by treatment with 5-aza-2'-deoxycytidine or lentiviral expression of cDNA, was performed to assess effects on cell viability, necrosis, and chemosensitization. RESULTS: Loss of RIPK3 expression was observed in 42/66 (63%) primary malignant mesotheliomas and malignant mesothelioma cell lines, and RT-PCR analysis demonstrated that downregulation occurs at the transcriptional level, consistent with epigenetic silencing. RIPK3-negative malignant mesothelioma cells treated with 5-aza-2'-deoxycytidine resulted in reexpression of RIPK3 and chemosensitization. Ectopic expression of RIPK3 also resulted in chemosensitization and led to necroptosis, the latter demonstrated by phosphorylation of downstream target MLKL and confirmed by rescue experiments. Mining of RIPK3 expression and survival outcomes among patients with malignant mesothelioma available from The Cancer Genome Atlas repository revealed that promoter methylation of RIPK3 is associated with reduced RIPK3 expression and poor prognosis. CONCLUSIONS: These data suggest that RIPK3 acts as a tumor suppressor in malignant mesothelioma by triggering necroptosis and that epigenetic silencing of RIPK3 by DNA methylation impairs necroptosis and contributes to chemoresistance and poor survival in this incurable disease.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética , Mesotelioma Maligno/tratamento farmacológico , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Masculino , Mesotelioma Maligno/genética , Mesotelioma Maligno/mortalidade , Mesotelioma Maligno/patologia , Camundongos , Pessoa de Meia-Idade , Necroptose/genética , Regiões Promotoras Genéticas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
As the 21st century uncovers ever-increasing volumes of asbestos and asbestos-contaminated waste, we need a new way to stop 'grandfather's problem' from becoming that of our future generations. The production of inexpensive, mechanically strong, heat resistant building materials containing asbestos has inevitably led to its use in many public and residential buildings globally. It is therefore not surprising that since the asbestos boom in the 1970s, some 30 years later, the true extent of this hidden danger was exposed. Yet, this severely toxic material continues to be produced and used in some countries, and in others the disposal options for historic uses - generally landfill - are at best unwieldy and at worst insecure. We illustrate the global scale of the asbestos problem via three case studies which describe various removal and/or end disposal issues. These case studies from both industrialised and island nations demonstrate the potential for the generation of massive amounts of asbestos contaminated soil. In each case, the final outcome of the project was influenced by factors such as cost and land availability, both increasing issues, worldwide. The reduction in the generation of asbestos containing materials will not absolve us from the necessity of handling and disposal of contaminated land. Waste treatment which relies on physico-chemical processes is expensive and does not contribute to a circular model economy ideal. Although asbestos is a mineral substance, there are naturally occurring biological-mediated processes capable of degradation (such as bioweathering). Therefore, low energy options, such as bioremediation, for the treatment for asbestos contaminated soils are worth exploring. We outline evidence pointing to the ability of microbe and plant communities to remove from asbestos the iron that contributes to its carcinogenicity. Finally, we describe the potential for a novel concept of creating ecosystems over asbestos landfills ('activated landfills') that utilize nature's chelating ability to degrade this toxic product effectively.
RESUMO
Pleural malignant mesothelioma is a therapy-resistant cancer affecting the serosal lining of the thoracic cavity. Mutations/deletions of BAP1, CDKN2A, and NF2 are the most frequent genetic lesions in human malignant mesothelioma. We introduced various combinations of these deletions in the pleura of conditional knockout (CKO) mice, focusing on the contribution of Bap1 loss. While homozygous CKO of Bap1, Cdkn2a, or Nf2 alone gave rise to few or no malignant mesotheliomas, inactivation of Bap1 cooperated with loss of either Nf2 or Cdkn2a to drive development of malignant mesothelioma in approximately 20% of double-CKO mice, and a high incidence (22/26, 85%) of malignant mesotheliomas was observed in Bap1;Nf2;Cdkn2a (triple)-CKO mice. Malignant mesothelioma onset was rapid in triple-CKO mice, with a median survival of only 12 weeks, and malignant mesotheliomas from these mice were consistently high-grade and invasive. Adenoviral-Cre treatment of normal mesothelial cells from Bap1;Nf2;Cdkn2a CKO mice, but not from mice with knockout of one or any two of these genes, resulted in robust spheroid formation in vitro, suggesting that mesothelial cells from Bap1;Nf2;Cdkn2a mice have stem cell-like potential. RNA-seq analysis of malignant mesotheliomas from triple-CKO mice revealed enrichment of genes transcriptionally regulated by the polycomb repressive complex 2 (PRC2) and others previously implicated in known Bap1-related cellular processes. These data demonstrate that somatic inactivation of Bap1, Nf2, and Cdkn2a results in rapid, aggressive malignant mesotheliomas, and that deletion of Bap1 contributes to tumor development, in part, by loss of PRC2-mediated repression of tumorigenic target genes and by acquisition of stem cell potential, suggesting a potential avenue for therapeutic intervention. SIGNIFICANCE: Combinatorial deletions of Bap1, Nf2, and Cdkn2a result in aggressive mesotheliomas, with Bap1 loss contributing to tumorigenesis by circumventing PRC2-mediated repression of oncogenic target genes.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Neurofibromina 2/genética , Neoplasias Pleurais/patologia , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Animais , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Mesotelioma/genética , Mesotelioma Maligno , Camundongos Knockout , Neurofibromina 2/metabolismo , Neoplasias Pleurais/genética , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
Malignant mesothelioma (MM) is a therapy-resistant cancer arising primarily from the lining of the pleural and peritoneal cavities. The most frequently altered genes in human MM are cyclin-dependent kinase inhibitor 2A (CDKN2A), which encodes components of the p53 (p14ARF) and RB (p16INK4A) pathways, BRCA1-associated protein 1 (BAP1), and neurofibromatosis 2 (NF2). Furthermore, the p53 gene (TP53) itself is mutated in ~15% of MMs. In many MMs, the PI3K-PTEN-AKT-mTOR signaling node is hyperactivated, which contributes to tumor cell survival and therapeutic resistance. Here, we demonstrate that the inactivation of both Tp53 and Pten in the mouse mesothelium is sufficient to rapidly drive aggressive MMs. PtenL/L ;Tp53L/L mice injected intraperitoneally or intrapleurally with adenovirus-expressing Cre recombinase developed high rates of peritoneal and pleural MMs (92% of mice with a median latency of 9.4 weeks and 56% of mice with a median latency of 19.3 weeks, respectively). MM cells from these mice showed consistent activation of Akt-mTor signaling, chromosome breakage or aneuploidy, and upregulation of Myc; occasional downregulation of Bap1 was also observed. Collectively, these findings suggest that when Pten and Tp53 are lost in combination in mesothelial cells, DNA damage is not adequately repaired and genomic instability is widespread, whereas the activation of Akt due to Pten loss protects genomically damaged cells from apoptosis, thereby increasing the likelihood of tumor formation. Additionally, the mining of an online dataset (The Cancer Genome Atlas) revealed codeletions of PTEN and TP53 and/or CDKN2A/p14ARF in ~25% of human MMs, indicating that cooperative losses of these genes contribute to the development of a significant proportion of these aggressive neoplasms and suggesting key target pathways for therapeutic intervention.
Assuntos
Neoplasias Pulmonares/genética , Mesotelioma/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias Pleurais/genética , Proteína Supressora de Tumor p53/genética , Animais , Carcinogênese/genética , Proliferação de Células/genética , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Mesotelioma Maligno , Camundongos , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/patologia , Neoplasias Pleurais/patologia , Transdução de Sinais , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genéticaRESUMO
Tumor suppressor genes and their effector pathways have been identified for many dominantly heritable cancers, enabling efforts to intervene early in the course of disease. Our approach on the subject of early intervention was to investigate gene expression patterns of morphologically normal "one-hit" cells before they become hemizygous or homozygous for the inherited mutant gene which is usually required for tumor formation. Here, we studied histologically non-transformed renal epithelial cells from patients with inherited disorders that predispose to renal tumors, including von Hippel-Lindau (VHL) disease and Tuberous Sclerosis (TSC). As controls, we studied histologically normal cells from non-cancerous renal epithelium of patients with sporadic clear cell renal cell carcinoma (ccRCC). Gene expression analyses of VHLmut/wt or TSC1/2mut/wt versus wild-type (WT) cells revealed transcriptomic alterations previously implicated in the transition to precancerous renal lesions. For example, the gene expression changes in VHLmut/wt cells were consistent with activation of the hypoxia response, associated, in part, with the "Warburg effect". Knockdown of any remaining VHL mRNA using shRNA induced secondary expression changes, such as activation of NFκB and interferon pathways, that are fundamentally important in the development of RCC. We posit that this is a general pattern of hereditary cancer predisposition, wherein haploinsufficiency for VHL or TSC1/2, or potentially other tumor susceptibility genes, is sufficient to promote development of early lesions, while cancer results from inactivation of the remaining normal allele. The gene expression changes identified here are related to the metabolic basis of renal cancer and may constitute suitable targets for early intervention.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Predisposição Genética para Doença/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Haploinsuficiência , Heterozigoto , Humanos , Immunoblotting , Neoplasias Renais/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , TranscriptomaRESUMO
Exposure to asbestos is causally associated with the development of malignant mesothelioma, a cancer of cells lining the internal body cavities. Malignant mesothelioma is an aggressive cancer resistant to all current therapies. Once inhaled or ingested, asbestos causes inflammation in and around tissues that come in contact with these carcinogenic fibers. Recent studies suggest that inflammation is a major contributing factor in the development of many types of cancer, including malignant mesothelioma. The NALP3/NLRP3 inflammasome, including the component ASC, is thought to be an important mediator of inflammation in cells that sense extracellular insults, such as asbestos, and activate a signaling cascade resulting in release of mature IL1ß and recruitment of inflammatory cells. To determine if inflammasome-mediated inflammation contributes to asbestos-induced malignant mesothelioma, we chronically exposed Asc-deficient mice and wild-type littermates to asbestos and evaluated differences in tumor incidence and latency. The Asc-deficient mice showed significantly delayed tumor onset and reduced malignant mesothelioma incidence compared with wild-type animals. We also tested whether inflammation-related release of IL1ß contributes to tumor development in an accelerated mouse model of asbestos-induced malignant mesothelioma. Nf2(+/-);Cdkn2a(+/-) mice exposed to asbestos in the presence of anakinra, an IL1 receptor (IL1R) antagonist, showed a marked delay in the median time of malignant mesothelioma onset compared with similarly exposed mice given vehicle control (33.1 weeks vs. 22.6 weeks, respectively). Collectively, these studies provide evidence for a link between inflammation-related IL1ß/IL1R signaling and the development of asbestos-induced malignant mesothelioma. Furthermore, these findings provide rationale for chemoprevention strategies targeting IL1ß/IL1R signaling in high-risk, asbestos-exposed populations. Cancer Prev Res; 9(5); 406-14. ©2016 AACR.
Assuntos
Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/metabolismo , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Receptores de Interleucina-1/metabolismo , Animais , Proteínas Reguladoras de Apoptose/deficiência , Amianto/efeitos adversos , Proteínas Adaptadoras de Sinalização CARD , Modelos Animais de Doenças , Humanos , Immunoblotting , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Mesotelioma Maligno , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologiaRESUMO
Individuals harboring inherited heterozygous germline mutations in BAP1 are predisposed to a range of benign and malignant tumor types, including malignant mesothelioma, melanoma, and kidney carcinoma. However, evidence to support a tumor-suppressive role for BAP1 in cancer remains contradictory. To test experimentally whether BAP1 behaves as a tumor suppressor, we monitored spontaneous tumor development in three different mouse models with germline heterozygous mutations in Bap1, including two models in which the knock-in mutations are identical to those reported in human BAP1 cancer syndrome families. We observed spontaneous malignant tumors in 54 of 93 Bap1-mutant mice (58%) versus 4 of 43 (9%) wild-type littermates. All three Bap1-mutant models exhibited a high incidence and similar spectrum of neoplasms, including ovarian sex cord stromal tumors, lung and mammary carcinomas, and spindle cell tumors. Notably, we also observed malignant mesotheliomas in two Bap1-mutant mice, but not in any wild-type animals. We further confirmed that the remaining wild-type Bap1 allele was lost in both spontaneous ovarian tumors and mesotheliomas, resulting in the loss of Bap1 expression. Additional studies revealed that asbestos exposure induced a highly significant increase in the incidence of aggressive mesotheliomas in the two mouse models carrying clinically relevant Bap1 mutations compared with asbestos-exposed wild-type littermates. Collectively, these findings provide genetic evidence that Bap1 is a bona fide tumor suppressor gene and offer key insights into the contribution of carcinogen exposure to enhanced cancer susceptibility. Cancer Res; 76(9); 2836-44. ©2016 AACR.
Assuntos
Genes Supressores de Tumor , Mutação em Linhagem Germinativa , Síndromes Neoplásicas Hereditárias , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Animais , Hibridização Genômica Comparativa , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Predisposição Genética para Doença/genética , Genótipo , Heterozigoto , Imuno-Histoquímica , Microdissecção e Captura a Laser , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: Sarcomatoid changes in renal cell carcinoma are associated with a poor prognosis. The identification of genetic alterations that drive this aggressive phenotype could aid in the development of more effective targeted therapies. In this study we aimed to pinpoint unique copy number alterations in sarcomatoid renal cell carcinoma compared to classical renal cell carcinoma subtypes. MATERIALS AND METHODS: Genomic copy number analysis was performed using single nucleotide polymorphism based microarrays on tissue extracted from the tumors of 81 patients who underwent renal mass excision, including 17 with sarcomatoid renal cell carcinoma. RESULTS: Sarcomatoid renal cell carcinoma showed a significantly higher number of copy number alterations than clear cell, papillary and chromophobe renal cell carcinoma (mean 18.0 vs 5.8, 6.5 and 7.2, respectively, p <0.0001). Copy number losses of chromosome arms 9q, 15q, 18p/q and 22q, and gains of 1q and 8q occurred in a significantly higher proportion of sarcomatoid renal cell carcinomas than in the other 3 histologies. Patients with sarcomatoid renal cell carcinoma demonstrated significantly worse overall survival compared to those without that condition on Kaplan-Meier analysis (p = 0.0001). Patients with 9 or more copy number alterations also demonstrated significantly worse overall survival than those with fewer than 9 copy number alterations (p = 0.004). CONCLUSIONS: Sarcomatoid changes in renal cell carcinoma are associated with a high rate of chromosomal imbalances with losses of 9q, 15q, 18p/q and 22q, and gains of 1q and 8q occurring at significantly higher frequencies in comparison to nonsarcomatoid renal cell carcinoma. Identifying candidate driver genes or tumor suppressor loci in these chromosomal regions may help identify targets for future therapies.
Assuntos
Carcinoma de Células Renais/genética , Variações do Número de Cópias de DNA , Neoplasias Renais/genética , Carcinoma de Células Renais/mortalidade , Predisposição Genética para Doença , Humanos , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Análise de Sobrevida , Análise Serial de Tecidos/métodosRESUMO
Malignant mesothelioma (MM), linked to asbestos exposure, is a highly lethal form of thoracic cancer with a long latency period, high mortality and poor treatment options. Chronic inflammation and oxidative tissue damage caused by asbestos fibers are linked to MM development. Flaxseed lignans, enriched in secoisolariciresinol diglucoside (SDG), have antioxidant, anti-inflammatory and cancer chemopreventive properties. As a prelude to chronic chemoprevention studies for MM development, we tested the ability of flaxseed lignan component (FLC) to prevent acute asbestos-induced inflammation in MM-prone Nf2(+/mu) mice. Mice (n = 16-17 per group) were placed on control (CTL) or FLC-supplemented diets initiated 7 days prior to a single intraperitoneal bolus of 400 µg of crocidolite asbestos. Three days post asbestos exposure, mice were evaluated for abdominal inflammation, proinflammatory/profibrogenic cytokine release, WBC gene expression changes and oxidative and nitrosative stress in peritoneal lavage fluid (PLF). Asbestos-exposed mice fed CTL diet developed acute inflammation, with significant (P < 0.0001) elevations in WBCs and proinflammatory/profibrogenic cytokines (IL-1ß, IL-6, TNFα, HMGB1 and active TGFß1) relative to baseline (BL) levels. Alternatively, asbestos-exposed FLC-fed mice had a significant (P < 0.0001) decrease in PLF WBCs and proinflammatory/profibrogenic cytokine levels relative to CTL-fed mice. Importantly, PLF WBC gene expression of cytokines (IL-1ß, IL-6, TNFα, HMGB1 and TGFß1) and cytokine receptors (TNFαR1 and TGFßR1) were also downregulated by FLC. FLC also significantly (P < 0.0001) blunted asbestos-induced nitrosative and oxidative stress. FLC reduces acute asbestos-induced peritoneal inflammation, nitrosative and oxidative stress and may thus prove to be a promising agent in the chemoprevention of MM.
Assuntos
Asbesto Crocidolita/toxicidade , Butileno Glicóis/administração & dosagem , Glucosídeos/administração & dosagem , Inflamação/patologia , Lignanas/administração & dosagem , Peritônio/patologia , Animais , Antioxidantes/farmacologia , Cromatografia Líquida , Dieta , Suplementos Nutricionais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Linho , Mesotelioma/patologia , Camundongos , Camundongos Mutantes , Estresse Oxidativo/efeitos dos fármacos , Lavagem Peritoneal , Peritônio/efeitos dos fármacos , Lesões Pré-Cancerosas/tratamento farmacológico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes , Espectrometria de Massas em Tandem , TranscriptomaRESUMO
Malignant mesotheliomas are highly aggressive tumors usually caused by exposure to asbestos. Germline-inactivating mutations of BAP1 predispose to mesothelioma and certain other cancers. However, why mesothelioma is the predominate malignancy in some BAP1 families and not others, and whether exposure to asbestos is required for development of mesothelioma in BAP1 mutation carriers are not known. To address these questions experimentally, we generated a Bap1(+/-) knockout mouse model to assess its susceptibility to mesothelioma upon chronic exposure to asbestos. Bap1(+/-) mice exhibited a significantly higher incidence of asbestos-induced mesothelioma than wild-type (WT) littermates (73% vs. 32%, respectively). Furthermore, mesotheliomas arose at an accelerated rate in Bap1(+/-) mice than in WT animals (median survival, 43 weeks vs. 55 weeks after initial exposure, respectively) and showed increased invasiveness and proliferation. No spontaneous mesotheliomas were seen in unexposed Bap1(+/-) mice followed for up to 87 weeks of age. Mesothelioma cells from Bap1(+/-) mice showed biallelic inactivation of Bap1, consistent with its proposed role as a recessive cancer susceptibility gene. Unlike in WT mice, mesotheliomas from Bap1(+/-) mice did not require homozygous loss of Cdkn2a. However, normal mesothelial cells and mesothelioma cells from Bap1(+/-) mice showed downregulation of Rb through a p16(Ink4a)-independent mechanism, suggesting that predisposition of Bap1(+/-) mice to mesothelioma may be facilitated, in part, by cooperation between Bap1 and Rb. Drawing parallels to human disease, these unbiased genetic findings indicate that BAP1 mutation carriers are predisposed to the tumorigenic effects of asbestos and suggest that high penetrance of mesothelioma requires such environmental exposure.
Assuntos
Amianto/toxicidade , Mutação em Linhagem Germinativa , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/genética , Mesotelioma/etiologia , Mesotelioma/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Animais , Modelos Animais de Doenças , Epigenômica , Feminino , Predisposição Genética para Doença , Genótipo , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Mesotelioma Maligno , Camundongos , Camundongos Knockout , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
The goal of targeted therapy is to match a selective drug with a genetic lesion that predicts for drug sensitivity. In a diverse panel of cancer cell lines, we found that the cells most sensitive to focal adhesion kinase (FAK) inhibition lack expression of the neurofibromatosis type 2 (NF2) tumor suppressor gene product, Merlin. Merlin expression is often lost in malignant pleural mesothelioma (MPM), an asbestos-induced aggressive cancer with limited treatment options. Our data demonstrate that low Merlin expression predicts for increased sensitivity of MPM cells to a FAK inhibitor, VS-4718, in vitro and in tumor xenograft models. Disruption of MPM cell-cell or cell-extracellular matrix (ECM) contacts with blocking antibodies suggests that weak cell-cell adhesions in Merlin-negative MPM cells underlie their greater dependence on cell-ECM-induced FAK signaling. This provides one explanation of why Merlin-negative cells are vulnerable to FAK inhibitor treatment. Furthermore, we validated aldehyde dehydrogenase as a marker of cancer stem cells (CSCs) in MPM, a cell population thought to mediate tumor relapse after chemotherapy. Whereas pemetrexed and cisplatin, standard-of-care agents for MPM, enrich for CSCs, FAK inhibitor treatment preferentially eliminates these cells. These preclinical results provide the rationale for a clinical trial in MPM patients using a FAK inhibitor as a single agent after first-line chemotherapy. With this design, the FAK inhibitor could potentially induce a more durable clinical response through reduction of CSCs along with a strong antitumor effect. Furthermore, our data suggest that patients with Merlin-negative tumors may especially benefit from FAK inhibitor treatment.
Assuntos
Antineoplásicos/farmacologia , Quinase 1 de Adesão Focal/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Mesotelioma/tratamento farmacológico , Neurofibromina 2/deficiência , Inibidores de Proteínas Quinases/farmacologia , Aldeído Desidrogenase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mesotelioma/enzimologia , Mesotelioma/genética , Mesotelioma/patologia , Mesotelioma Maligno , Camundongos , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Neurofibromina 2/genética , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Malignant mesothelioma is a highly aggressive, asbestos-related cancer frequently marked by mutations of both NF2 and CDKN2A. We demonstrate that germline knockout of one allele of each of these genes causes accelerated onset and progression of asbestos-induced malignant mesothelioma compared with asbestos-exposed Nf2(+/-) or wild-type mice. Ascites from some Nf2(+/-);Cdkn2a(+/-) mice exhibited large tumor spheroids, and tail vein injections of malignant mesothelioma cells established from these mice, but not from Nf2(+/-) or wild-type mice, produced numerous tumors in the lung, suggesting increased metastatic potential of tumor cells from Nf2(+/-);Cdkn2a(+/-) mice. Intraperitoneal injections of malignant mesothelioma cells derived from Nf2(+/-);Cdkn2a(+/-) mice into severe combined immunodeficient mice produced tumors that penetrated the diaphragm and pleural cavity and harbored increased cancer stem cells (CSC). Malignant mesothelioma cells from Nf2(+/-);Cdkn2a(+/-) mice stained positively for CSC markers and formed CSC spheroids in vitro more efficiently than counterparts from wild-type mice. Moreover, tumor cells from Nf2(+/-);Cdkn2a(+/-) mice showed elevated c-Met expression/activation, which was partly dependent on p53-mediated regulation of miR-34a and required for tumor migration/invasiveness and maintenance of the CSC population. Collectively, these studies demonstrate in vivo that inactivation of Nf2 and Cdkn2a cooperate to drive the development of highly aggressive malignant mesotheliomas characterized by enhanced tumor spreading capability and the presence of a CSC population associated with p53/miR-34a-dependent activation of c-Met. These findings suggest that cooperativity between losses of Nf2 and Cdkn2a plays a fundamental role in driving the highly aggressive tumorigenic phenotype considered to be a hallmark of malignant mesothelioma.
Assuntos
Proliferação de Células , Transformação Celular Neoplásica/genética , Genes Supressores de Tumor/fisiologia , Mesotelioma/genética , Células-Tronco Neoplásicas/fisiologia , Neoplasias Pleurais/genética , Animais , Amianto , Genes da Neurofibromatose 2/fisiologia , Genes p53/fisiologia , Mesotelioma/patologia , Camundongos , Camundongos SCID , Camundongos Transgênicos , MicroRNAs/fisiologia , Mutação , Invasividade Neoplásica , Células-Tronco Neoplásicas/patologia , Neoplasias Pleurais/patologia , Proteínas Proto-Oncogênicas c-met/fisiologia , Transdução de Sinais/genética , Células Tumorais CultivadasRESUMO
Group I p21-activated kinases (PAK) are important effectors of the small GTPases Rac and Cdc42, which regulate cell motility/migration, survival, proliferation, and gene transcription. Hyperactivation of these kinases have been reported in many tumor types, making PAKs attractive targets for therapeutic intervention. PAKs are activated by growth factor-mediated signaling and are negatively regulated by the tumor suppressor neurofibromatosis type 2 (NF2)/Merlin. Thus, tumors characterized by NF2 inactivation would be expected to show hyperactivated PAK signaling. On the basis of this rationale, we evaluated the status of PAK signaling in malignant mesothelioma, an aggressive neoplasm that is resistant to current therapies and shows frequent inactivation of NF2. We show that group I PAKs are activated in most mesotheliomas and mesothelioma cell lines and that genetic or pharmacologic inhibition of PAKs is sufficient to inhibit mesothelioma cell proliferation and survival. We also identify downstream effectors and signaling pathways that may contribute mechanistically to PAK-related tumorigenesis. Specifically, we show that inhibition of PAK results in attenuation of AKT and Raf-MAPK signaling and decreased tumor cell viability. Collectively, these data suggest that pharmacologic inhibition of group I PAKs may have therapeutic efficacy in tumors characterized by PAK activation.
Assuntos
Dissulfetos/farmacologia , Regulação Neoplásica da Expressão Gênica/genética , Mesotelioma/genética , Naftóis/farmacologia , Neurofibromina 2/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Técnicas de Silenciamento de Genes , Humanos , Mesotelioma/tratamento farmacológico , Mesotelioma/metabolismo , Camundongos , Neurofibromina 2/antagonistas & inibidores , Neurofibromina 2/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismoRESUMO
Signaling between the epithelium and stromal cells is crucial for growth, differentiation, and repair of the epithelium. Although the retinoblastoma protein (Rb) is known to regulate the growth of keratinocytes in a cell-autonomous manner, here we describe a function of Rb in the stromal compartment. We find that Rb depletion in fibroblasts leads to inhibition of differentiation and enhanced proliferation of the epithelium. Analysis of conditioned medium identified that keratinocyte growth factor (KGF) levels were elevated following Rb depletion. These findings were also observed with organotypic co-cultures. Treatment of keratinocytes with KGF inhibited differentiation and enhanced keratinocyte proliferation, whereas reduction of KGF levels in Rb-depleted fibroblasts was able to restore expression of differentiation markers. Our findings suggest a crucial role for dermal fibroblasts in regulating the differentiation and proliferation of keratinocytes, and we demonstrate a role for stromal Rb in this cross-talk.
Assuntos
Células Epidérmicas , Queratinócitos/citologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Células Estromais/citologia , Diferenciação Celular/fisiologia , Linhagem Celular Transformada , Proliferação de Células , Células Cultivadas , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Prepúcio do Pênis/citologia , Humanos , Interleucina-1alfa/metabolismo , Interleucina-1alfa/farmacologia , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Queratinócitos/metabolismo , Masculino , Boca/citologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células Estromais/metabolismoRESUMO
The CDKN2A/ARF locus encompasses overlapping tumor suppressor genes p16(INK4A) and p14(ARF), which are frequently co-deleted in human malignant mesothelioma (MM). The importance of p16(INK4A) loss in human cancer is well established, but the relative significance of p14(ARF) loss has been debated. The tumor predisposition of mice singly deficient for either Ink4a or Arf, due to targeting of exons 1α or 1ß, respectively, supports the idea that both play significant and nonredundant roles in suppressing spontaneous tumors. To further test this notion, we exposed Ink4a(+/-) and Arf(+/-) mice to asbestos, the major cause of MM. Asbestos-treated Ink4a(+/-) and Arf(+/-) mice showed increased incidence and shorter latency of MM relative to wild-type littermates. MMs from Ink4a(+/-) mice exhibited biallelic inactivation of Ink4a, loss of Arf or p53 expression and frequent loss of p15(Ink4b). In contrast, MMs from Arf(+/-) mice exhibited loss of Arf expression, but did not require loss of Ink4a or Ink4b. Mice doubly deficient for Ink4a and Arf, due to deletion of Cdkn2a/Arf exon 2, showed accelerated asbestos-induced MM formation relative to mice deficient for Ink4a or Arf alone, and MMs exhibited biallelic loss of both tumor suppressor genes. The tumor suppressor function of Arf in MM was p53-independent, since MMs with loss of Arf retained functional p53. Collectively, these in vivo data indicate that both CDKN2A/ARF gene products suppress asbestos carcinogenicity. Furthermore, while inactivation of Arf appears to be crucial for MM pathogenesis, the inactivation of both p16(Ink4a) and p19(Arf) cooperate to accelerate asbestos-induced tumorigenesis.
Assuntos
Amianto/efeitos adversos , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Loci Gênicos/genética , Mesotelioma/genética , Mesotelioma/patologia , Lesões Pré-Cancerosas/patologia , Proteína Supressora de Tumor p14ARF/deficiência , Alelos , Animais , Cromossomos de Mamíferos/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inativação Gênica , Humanos , Camundongos , Lesões Pré-Cancerosas/genética , Proteína Supressora de Tumor p14ARF/metabolismoRESUMO
The distal-less homeobox gene (dlx) 5 encodes a transcription factor that controls jaw formation and appendage differentiation during embryonic development. We had previously found that Dlx5 is overexpressed in an Akt2 transgenic model of T-cell lymphoma. To investigate if DLX5 is involved in human cancer, we screened its expression in the NCI 60 cancer cell line panel. DLX5 was frequently upregulated in cell lines derived from several tumor types, including ovarian cancer. We next validated its upregulation in primary ovarian cancer specimens. Stable knockdown of DLX5 by lentivirus-mediated transduction of short hairpin RNA (shRNA) resulted in reduced proliferation of ovarian cancer cells due to inhibition of cell cycle progression in connection with the downregulation of cyclins A, B1, D1, D2, and E, and decreased phosphorylation of AKT. Cell proliferation resumed following introduction of a DLX5 cDNA harboring wobbled mutations at the shRNA-targeting sites. Cell proliferation was also rescued by transduction of a constitutively active form of AKT. Intriguingly, downregulation of IRS-2 and MET contributed to the suppression of AKT signaling. Moreover, DLX5 was found to directly bind to the IRS-2 promoter and augmented its transcription. Knockdown of DLX5 in xenografts of human ovarian cancer cells resulted in markedly diminished tumor size. In addition, DLX5 was found to cooperate with HRAS in the transformation of human ovarian surface epithelial cells. Together, these data suggest that DLX5 plays a significant role in the pathogenesis of some ovarian cancers.
Assuntos
Proliferação de Células , Proteínas de Homeodomínio/genética , Proteínas Substratos do Receptor de Insulina/genética , Neoplasias Ovarianas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Fatores de Transcrição/genética , Animais , Western Blotting , Ciclo Celular , Linhagem Celular Tumoral , Células Cultivadas , Ciclinas/genética , Ciclinas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , Camundongos SCID , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Carga Tumoral , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Malignant mesothelioma (MM) is a highly aggressive cancer that is refractory to all current chemotherapeutic regimens. Therefore, uncovering new rational therapeutic targets is imperative in the field. Tyrosine kinase signaling pathways are aberrantly activated in many human cancers and are currently being targeted for chemotherapeutic intervention. Thus, we sought to identify tyrosine kinases hyperactivated in MM. An unbiased phosphotyrosine proteomic screen was employed to identify tyrosine kinases activated in human MM cell lines. From this screen, we have identified novel signaling molecules, such as JAK1, STAT1, cortactin (CTTN), FER, p130Cas (BCAR1), SRC and FYN as tyrosine phosphorylated in human MM cell lines. Additionally, STAT1 and SRC family kinases (SFK) were confirmed to be active in primary MM specimens. We also confirmed that known signal transduction pathways previously implicated in MM, such as EGFR and MET signaling axes, are co-activated in the majority of human MM specimens and cell lines tested. EGFR, MET, and SFK appear to be co-activated in a significant proportion of MM cell lines, and dual inhibition of these kinases was demonstrated to be more efficacious for inhibiting MM cell viability and downstream effector signaling than inhibition of a single tyrosine kinase. Consequently, these data suggest that TKI mono-therapy may not represent an efficacious strategy for the treatment of MM, due to multiple tyrosine kinases potentially signaling redundantly to cellular pathways involved in tumor cell survival and proliferation.
RESUMO
The E6 and E7 oncoproteins of high-risk human papillomaviruses (HPVs) are together sufficient to cause cellular transformation. Nucleophosmin (NPM) was identified as a protein with increased levels in two-dimensional (2-D) gel analysis of human foreskin keratinocytes (HFKs) expressing E7 following methylcellulose-induced differentiation. Analysis of NPM expression in E7-expressing cells and E6- and E7-expressing cells in culture and in organotypic rafts confirmed the increased levels observed in 2-D gel analysis. The elevated expression of NPM was determined to be posttranscriptional and was attributed to increased v-akt murine thymoma viral oncogene (AKT) activity in the E6- and E7-expressing cells. Depletion of NPM caused a reduction in the replicative capacity of E7- and E6/E7-expressing HFKs and an increase in markers of differentiation. Also, the p53 and pRb tumor suppressor levels are increased with the knockdown of NPM in E6/E7-expressing cells, and, interestingly, p14(ARF) is relocalized from the nucleolus to the nucleoplasm and cytoplasm in these cells. The results show for the first time that NPM is required for the proliferation and inhibition of differentiation observed in HPV E6- and E7-expressing primary cells.