RESUMO
Whereas aberrant activation of canonical Wnt/ß-catenin signaling underlies the majority of colorectal cancer cases, the contribution of non-canonical Wnt signaling is unclear. As enhanced expression of the most extensively studied non-canonical Wnt ligand WNT5A is observed in various diseases including colon cancer, WNT5A is gaining attention nowadays. Numerous in vitro studies suggest modulating capacities of WNT5A on proliferation, differentiation, migration and invasion, affecting tumor and non-mutant cells. However, a possible contribution of WNT5A to colorectal cancer remains to be elucidated. We have analyzed WNT5A expression in colorectal cancer profiling data sets, altered WNT5A expression in colon cancer cells and used our inducible Wnt5a transgenic mouse model to gain more insight into the role of WNT5A in intestinal cancer. We observed that increased WNT5A expression is associated with poor prognosis of colorectal cancer patients. WNT5A knockdown in human colon cancer cells caused reduced directional migration, deregulated focal adhesion site formation and reduced invasion, whereas Wnt5a administration promoted the directional migration of colon cancer cells. Despite these observed protumorigenic activities of WNT5A, the induction of Wnt5a expression in intestinal tumors of Apc1638N mice was not sufficient to augment malignancy or metastasis by itself. In conclusion, WNT5A promotes adhesion sites to form in a focal fashion and promotes the directional migration and invasion of colon cancer cells. Although these activities appear insufficient by themselves to augment malignancy or metastasis in Apc1638N mice, they might explain the poor colon cancer prognosis associated with enhanced WNT5A expression.
Assuntos
Proteína da Polipose Adenomatosa do Colo/fisiologia , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/patologia , Neoplasias do Colo/patologia , Intestinos/patologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Animais , Apoptose , Western Blotting , Adesão Celular , Transformação Celular Neoplásica/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Adesões Focais , Humanos , Técnicas Imunoenzimáticas , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Invasividade Neoplásica , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteínas Wnt/genética , Proteína Wnt-5aRESUMO
Peutz-Jeghers syndrome (PJS) is caused by mutations in the LKB1 gene. It is characterized by gastrointestinal polyposis and an increased cancer risk, mainly in the gastrointestinal tract. Mechanisms of PJS-associated carcinogenesis are unclear. We investigated the involvement of candidate genes and molecular pathways in PJS-associated gastrointestinal cancers and dysplastic hamartomas. Cases were selected from the Dutch PJS cohort. Available tissue was immunostained for phospho-S6, ß-catenin, P53 and SMAD4. DNA was isolated from carcinoma tissue and dysplastic and non-dysplastic areas of hamartomas specifically. Mutation analyses were done for BRAF, KRAS and P53, and loss of heterozygosity (LOH) analyses for LKB1 and P53. Twenty-four of 144 patients (17%) developed 26 gastrointestinal malignancies at a median age of 49 years (interquartile range: 35-60). Eleven of 792 hamartomas (1.4%) of 9 patients were classified as dysplastic. LOH of LKB1 was detected in three of six (50%) carcinomas and in the dysplastic part of three of five (60%) hamartomas. Aberrant P53 expression was observed in 8 of 15 (53%) carcinomas. Six carcinomas with P53 overexpression harboured a P53 mutation, with loss of the remaining wild-type allele in four. Two hamartomas showing P53 overexpression in high-grade dysplastic foci harboured a P53 mutation with LOH. Loss of nuclear SMAD4 was observed in high-grade dysplastic foci of two of four (50%) hamartomas, in contrast to low-grade dysplastic foci (0/4) and non-dysplastic epithelium. Our findings suggest a role for mutant P53 in PJS-associated gastrointestinal carcinogenesis. Inactivation of transforming growth factor-ß/bone morphogenetic protein signalling and complete loss of LKB1 might be involved in dysplastic transformation of gastrointestinal hamartomas specifically.
Assuntos
Neoplasias Gastrointestinais/genética , Regulação Neoplásica da Expressão Gênica , Hamartoma/genética , Síndrome de Peutz-Jeghers/genética , Quinases Proteína-Quinases Ativadas por AMP , Adulto , Alelos , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Neoplasias Gastrointestinais/patologia , Mutação em Linhagem Germinativa , Hamartoma/patologia , Humanos , Imuno-Histoquímica , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Países Baixos , Linhagem , Síndrome de Peutz-Jeghers/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Peutz-Jeghers syndrome (PJS), caused by germ-line mutations in LKB1, is characterized by the development of hamartomatous polyps in the gastrointestinal (GI) tract. McCune Albright syndrome (MAS), caused by somatic activating mutations in GNAS, presents with cutaneous, skeletal, and endocrine manifestations. Recently, hamartomatous GI polyps with histological features similar to those in PJS were observed in MAS patients, suggesting a role for GNAS in the pathogenesis of PJS. This study reports the first somatic GNAS mutation analysis in GI tumours of PJS patients. No mutations were observed, suggesting that GNAS is not involved in the pathogenesis of GI tumours in PJS.
Assuntos
Adenocarcinoma/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Neoplasias Gastrointestinais/genética , Hamartoma/genética , Mutação/genética , Síndrome de Peutz-Jeghers/genética , Adenocarcinoma/patologia , Cromograninas , Estudos de Coortes , Análise Mutacional de DNA , Neoplasias Gastrointestinais/patologia , Hamartoma/patologia , Humanos , Síndrome de Peutz-Jeghers/patologia , Reação em Cadeia da Polimerase , PrognósticoRESUMO
BACKGROUND: Although Peutz-Jeghers syndrome (PJS) is known to be associated with pancreatic cancer (PC), estimates of this risk differ widely. This hampers counselling of patients and implementation of surveillance strategies. We therefore aimed to determine the PC risk in a large cohort of Dutch PJS patients. METHODS: PJS was defined by diagnostic criteria recommended by the WHO, a proven LKB1 mutation, or both. All patients with a presumptive diagnosis of pancreatic, ampullary or distal bile duct cancer were identified. Cases were reviewed clinically, radiologically and immunohistochemically. Cumulative PC risks were calculated by Kaplan-Meier analysis and relative risks by Poisson regression analysis. RESULTS: We included 144 PJS patients (49% male) from 61 families (5640 person years follow-up). Seven (5%) patients developed PC at a median age of 54 years. Four patients (3%) were diagnosed with distal bile duct (n=2) or ampullary cancer (n=2) at a median age of 55 years. The cumulative risk for PC was 26% (95% CI 4% to 47%) at age 70 years and relative risk was 76 (95% CI 36 to 160; p<0.001). The cumulative risk for pancreatico-biliary cancer was 32% (95% CI 11% to 52%) at age 70 years, with a relative risk of 96 (95% CI 53 to 174; p<0.001). CONCLUSIONS: PJS patients have a highly increased risk for pancreatico-biliary cancer. Therefore, patients are eligible for surveillance within well defined research programmes to establish the benefit of such surveillance.
Assuntos
Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/epidemiologia , Síndrome de Peutz-Jeghers/complicações , Risco , Adulto , Estudos de Coortes , Bases de Dados Factuais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Síndrome de Peutz-Jeghers/epidemiologia , Vigilância da População , Adulto JovemRESUMO
In contrast to the majority of sporadic colorectal cancer which predominantly occur in the distal colon, most mismatch repair deficient tumours arise at the proximal side. At present, these regional preferences have not been explained properly. Recently, we have screened colorectal tumours for mutations in Wnt-related genes focusing specifically on colorectal location. Combining this analysis with published data, we propose a mechanism underlying the side-related preferences of colorectal cancers, based on the specific acquired genetic defects in ß-catenin signalling.
Assuntos
Neoplasias Colorretais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Neoplasias Encefálicas , Genes APC , Humanos , Dados de Sequência Molecular , Mutação , Síndromes Neoplásicas Hereditárias , Especificidade de Órgãos , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/fisiologiaRESUMO
Objective Deregulation of the Wnt signalling pathway by mutations in the Apc or ß-catenin genes underlies colorectal carcinogenesis. As a result, ß-catenin stabilises, translocates to the nucleus, and activates gene transcription. Intestinal tumours show a heterogeneous pattern of nuclear ß-catenin, with the highest levels observed at the invasion front. Activation of receptor tyrosine kinases in these tumour areas by growth factors expressed by surrounding stromal cells phosphorylate ß-catenin at tyrosine residues, which is thought to increase ß-catenin nuclear translocation and tumour invasiveness. This study investigates the relevance of ß-catenin tyrosine phosphorylation for Wnt signalling and intestinal tumorigenesis in vivo. Design A conditional knock-in mouse model was generated into which the phospho-mimicking Y654E modification in the endogenous ß-catenin gene was introduced. Results This study provided in vivo evidence that ß-catenin(E654) is characterised by reduced affinity for cadherins, increased signalling and strongly increased phosphorylation at serine 675 by protein kinase A (PKA). In addition, homozygosity for the ß-catenin(E654) targeted allele caused embryonic lethality, whereas heterozygosity predisposed to intestinal tumour development, and strongly enhanced Apc-driven intestinal tumour initiation associated with increased nuclear accumulation of ßcatenin. Surprisingly, the expression of ß-catenin(E654) did not affect histological grade or induce tumour invasiveness. Conclusions A thus far unknown mechanism was uncovered in which Y654 phosphorylation of ß-catenin facilitates additional phosphorylation at serine 675 by PKA. In addition, in contrast to the current belief that ß-catenin Y654 phosphorylation increases tumour progression to a more invasive phenotype, these results show that it rather increases tumour initiation by enhancing Wnt signalling.
Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas Wnt/fisiologia , beta Catenina/metabolismo , Adenoma/genética , Adenoma/metabolismo , Animais , Células COS , Caderinas/metabolismo , Membrana Celular/metabolismo , Transformação Celular Neoplásica/genética , Chlorocebus aethiops , Neoplasias Colorretais/genética , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Perda do Embrião/genética , Técnicas de Introdução de Genes , Genes APC , Genótipo , Heterozigoto , Homozigoto , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologiaRESUMO
In multiple endocrine neoplasia syndrome Type 2 (MEN2), medullary thyroid carcinoma (MTC) and pheochromocytoma (PC) are associated with hereditary activating germ-line mutations in the RET proto-oncogene. Also in a large percentage of sporadic MTCs and PCs, somatic RET mutations appear to be involved in tumor formation. In one single MEN2 family an extensive variety in disease expression may be observed, indicating that additional genetic events are responsible for progression of the disease towards a more aggressive phenotype. However, these additional mutations in both hereditary and sporadic MTC and PC development are largely unknown. Here, we show for the first time the presence of somatic mutations in the cell cycle regulator P18 in human RET-associated MTCs and PCs. Each of these mutations causes an amino acid substitution in the cyclin dependent kinase-interacting region of P18(INK4C). Since these mutations partly inhibited P18(INK4C) function and reduced its stability, our findings implicate P18 as a tumor suppressor gene involved in human MTC and PC development.
Assuntos
Carcinoma Medular/metabolismo , Inibidor de Quinase Dependente de Ciclina p18/biossíntese , Inibidor de Quinase Dependente de Ciclina p18/fisiologia , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Feocromocitoma/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Análise Mutacional de DNA , Progressão da Doença , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Proto-Oncogene Mas , Homologia de Sequência de AminoácidosRESUMO
Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominantly inherited tumor syndrome subclassified into three distinct syndromes: MEN 2A, MEN 2B and familial medullary thyroid carcinoma. In MEN 2 families, medullary thyroid carcinoma, pheochromocytomas and parathyroid adenomas occur with a variable frequency, also depending on the specific genetic defect involved. In 1993, the responsible MEN2 gene was identified. The genetic defect in these disorders involves the RET proto-oncogene on chromosome 10. The germline RET mutations result in a gain-of-function of the RET protein. Extensive studies on large families revealed that there is a strong genotype-phenotype correlation. In this review, guidelines for early diagnosis, including MEN2 gene mutation analysis, and treatment, including preventive surgery, periodic and clinical monitoring, have been formulated, enabling improvement of life expectancy and quality of life. Identification of the RET protein has also provided new insights into its function, and the specific pathways it effects involved in cell proliferation, migration, differentiation and survival. In the near future, identification of biological tumor markers will enable target-directed intervention and may prevent and/or delay progression of both primary and residual tumor growth.
RESUMO
Activating mutations in the RET proto-oncogene are associated with both familial and sporadic medullary thyroid carcinoma (MTC) development; however, the genetic mechanisms underlying MTC tumorigenesis remain largely unknown. Recently, we have identified somatic inactivating mutations in the cell cycle inhibitor gene P18 in human MTC, which coincided with activating RET mutations, suggesting a role for loss of P18 in combination with oncogenic RET in the multistep process of MTC development. Therefore, we crossed transgenic mice expressing oncogenic RET (RET2B) with mice lacking p18 (and p27, another cell cycle inhibitor) and monitored MTC development. RET2B;p18(+/-) mice and RET2B;p18(-/-) mice developed MTC with a highly increased incidence compared with their corresponding single mutant littermates. In addition, expression of oncogenic RET causes an earlier age of onset and larger MTCs in p18(-/-);p27(+/-) mice. In a subset of MTCs of RET2B;p18(+/-)(;p27(+/-)) mice, p18(Ink4c) expression was completely lost. This loss of p18(Ink4c) expression correlated with higher proliferation rates as well as with larger MTCs, indicating that loss of p18 in combination with oncogenic RET not only increases the risk for MTC development but also enhances MTC progression. Our data strongly indicate that oncogenic RET and loss of p18 cooperate in the multistep tumorigenesis of MTC.
Assuntos
Carcinoma Medular/genética , Carcinoma Medular/patologia , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Regulação Neoplásica da Expressão Gênica , Mutação , Proteínas Proto-Oncogênicas c-ret/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Animais , Calcitonina/metabolismo , Ciclo Celular , Transformação Celular Neoplásica , Progressão da Doença , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proto-Oncogene MasRESUMO
The RET receptor tyrosine kinase has essential roles in cell survival, differentiation, and proliferation. Oncogenic activation of RET causes the cancer syndrome multiple endocrine neoplasia type 2 (MEN 2) and is a frequent event in sporadic thyroid carcinomas. However, the molecular mechanisms underlying RET's potent transforming and mitogenic signals are still not clear. Here, we show that nuclear localization of beta-catenin is frequent in both thyroid tumors and their metastases from MEN 2 patients, suggesting a novel mechanism of RET-mediated function through the beta-catenin signaling pathway. We show that RET binds to, and tyrosine phosphorylates, beta-catenin and show that the interaction between RET and beta-catenin can be direct and independent of cytoplasmic kinases, such as SRC. As a result of RET-mediated tyrosine phosphorylation, beta-catenin escapes cytosolic down-regulation by the adenomatous polyposis coli/Axin/glycogen synthase kinase-3 complex and accumulates in the nucleus, where it can stimulate beta-catenin-specific transcriptional programs in a RET-dependent fashion. We show that down-regulation of beta-catenin activity decreases RET-mediated cell proliferation, colony formation, and tumor growth in nude mice. Together, our data show that a beta-catenin-RET kinase pathway is a critical contributor to the development and metastasis of human thyroid carcinoma.
Assuntos
Carcinoma/metabolismo , Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-ret/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , beta Catenina/metabolismo , Animais , Carcinoma/patologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Transdução de Sinais , Neoplasias da Glândula Tireoide/patologiaRESUMO
Little is known about the regulation of cell fate decisions that lead to the formation of five pairs of mammary placodes in the surface ectoderm of the mouse embryo. We have previously shown that fibroblast growth factor 10 (FGF10) is required for the formation of mammary placodes 1, 2, 3 and 5. Here, we have found that Fgf10 is expressed only in the somites underlying placodes 2 and 3, in gradients across and within these somites. To test whether somitic FGF10 is required for the formation of these two placodes, we analyzed a number of mutants with different perturbations of somitic Fgf10 gradients for the presence of WNT signals and ectodermal multilayering, markers for mammary line and placode formation. The mammary line is displaced dorsally, and formation of placode 3 is impaired in Pax3ILZ/ILZ mutants, which do not form ventral somitic buds. Mammary line formation is impaired and placode 3 is absent in Gli3Xt-J/Xt-J and hypomorphic Fgf10 mutants, in which the somitic Fgf10 gradient is shortened dorsally and less overall Fgf10 is expressed, respectively. Recombinant FGF10 rescued mammogenesis in Fgf10(-/-) and Gli3Xt-J/Xt-J flanks. We correlate increasing levels of somitic FGF10 with progressive maturation of the surface ectoderm, and show that full expression of somitic Fgf10, co-regulated by GLI3, is required for the anteroposterior pattern in which the flank ectoderm acquires a mammary epithelial identity. We propose that the intra-somitic Fgf10 gradient, together with ventral elongation of the somites, determines the correct dorsoventral position of mammary epithelium along the flank.
Assuntos
Padronização Corporal , Indução Embrionária , Epitélio , Fator 10 de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Glândulas Mamárias Animais , Proteínas do Tecido Nervoso/metabolismo , Somitos/metabolismo , Animais , Ectoderma/citologia , Ectoderma/fisiologia , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Estruturas Embrionárias/citologia , Estruturas Embrionárias/fisiologia , Epitélio/embriologia , Epitélio/fisiologia , Feminino , Fator 10 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Fatores de Transcrição Kruppel-Like/genética , Glândulas Mamárias Animais/anatomia & histologia , Glândulas Mamárias Animais/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Somitos/citologia , Proteína Gli3 com Dedos de ZincoRESUMO
Mammogenesis in rabbit, rat, and human begins with the formation of an elevated ectodermal ridge in the embryo. Attempts to demonstrate a morphologically or histologically equivalent mammary line in the mouse have yielded controversial results. We show here that a mammary line exists in the mouse embryo at embryonic day (E) 11.25 as a concise line of Wnt10b expression and a broader band of Wnt6 expression in the surface ectoderm, between the subaxillary and suprainguinal region of each flank. Additional streaks of Wnt10b expression in the axillary and inguinal region join the mammary line on the flank slightly later. Expression of Wnt10b and Wnt6 becomes restricted to the placodes within 1.5 days. The ectoderm of the mammary line is organized as a pseudostratified epithelium connecting the developing mammary placodes at around E11.5, whereas all other surface ectoderm is single-layered. Thus, the mammary line expressing Wnt10b defines a distinct ectodermal region that we consider the equivalent of the ectodermal ridge in, for example, rabbit. To date, the formation of the mammary line expressing Wnt10b is the earliest discernible ectodermal event in murine embryonic mammary gland development.