RESUMO
We report that ~1.8% of all mesothelioma patients and 4.9% of those younger than 55, carry rare germline variants of the BRCA1 associated RING domain 1 (BARD1) gene that were predicted to be damaging by computational analyses. We conducted functional assays, essential for accurate interpretation of missense variants, in primary fibroblasts that we established in tissue culture from a patient carrying the heterozygous BARD1V523A mutation. We found that these cells had genomic instability, reduced DNA repair, and impaired apoptosis. Investigating the underlying signaling pathways, we found that BARD1 forms a trimeric protein complex with p53 and SERCA2 that regulates calcium signaling and apoptosis. We validated these findings in BARD1-silenced primary human mesothelial cells exposed to asbestos. Our study elucidated mechanisms of BARD1 activity and revealed that heterozygous germline BARD1 mutations favor the development of mesothelioma and increase the susceptibility to asbestos carcinogenesis. These mesotheliomas are significantly less aggressive compared to mesotheliomas in asbestos workers.
Assuntos
Sinalização do Cálcio , Reparo do DNA , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Mesotelioma , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Humanos , Reparo do DNA/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Mesotelioma/genética , Sinalização do Cálcio/genética , Feminino , Masculino , Pessoa de Meia-Idade , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Fibroblastos/metabolismo , Amianto/toxicidade , Instabilidade GenômicaRESUMO
BRCA1 associated protein 1 (BAP1) is a ubiquitin C-terminal hydrolase that deubiquitinates histone H2AK119ub and other proteins and regulates the expression of multiple genes. The knockout of this tumor suppressor gene results in severe thymic atrophy, complete loss of the T cell lineage, and abnormal B cell development in mice. In the current study, we investigated in vitro effects of BAP1 knockout on cytokine and chemokine production using the human B-lymphoblast cell line TSCE5. We confirmed that knockout changed the production of innate immune-associated genes and their receptors. The CCL19, CCR7, CCL2, and CXCR5 genes associated with T and B cell migration were upregulated. Knockout cells producing high levels of CCL19 showed acceleration of actin polymerization, which is essential for cell migration. CD69, PTPRC, and TLR3 genes that activate inflammation were downregulated. The tumor necrosis factor ligand genes TNF, LTA, and TNFSF10 were downregulated by knockout. In knockout cells, TNFα production was strongly downregulated upon the addition of H2 O2 , but NF-κB in the basal condition and when TNFα was added was augmented, suggesting that these cells could respond to TNFα. These results indicated that BAP1 affects the expression of chemokines and cytokines, T and B cell migration, and activated inflammation associating with innate immunity.
Assuntos
Citocinas , Ubiquitina Tiolesterase , Humanos , Camundongos , Animais , Ubiquitina Tiolesterase/genética , Citocinas/genética , Camundongos Knockout , Quimiocinas/genética , Imunidade Inata , Proteínas Supressoras de Tumor/genéticaRESUMO
Rare biallelic BLM gene mutations cause Bloom syndrome. Whether BLM heterozygous germline mutations (BLM+/-) cause human cancer remains unclear. We sequenced the germline DNA of 155 mesothelioma patients (33 familial and 122 sporadic). We found 2 deleterious germline BLM+/- mutations within 2 of 33 families with multiple cases of mesothelioma, one from Turkey (c.569_570del; p.R191Kfs*4) and one from the United States (c.968A>G; p.K323R). Some of the relatives who inherited these mutations developed mesothelioma, while none with nonmutated BLM were affected. Furthermore, among 122 patients with sporadic mesothelioma treated at the US National Cancer Institute, 5 carried pathogenic germline BLM+/- mutations. Therefore, 7 of 155 apparently unrelated mesothelioma patients carried BLM+/- mutations, significantly higher (P = 6.7E-10) than the expected frequency in a general, unrelated population from the gnomAD database, and 2 of 7 carried the same missense pathogenic mutation c.968A>G (P = 0.0017 given a 0.00039 allele frequency). Experiments in primary mesothelial cells from Blm+/- mice and in primary human mesothelial cells in which we silenced BLM revealed that reduced BLM levels promote genomic instability while protecting from cell death and promoted TNF-α release. Blm+/- mice injected intraperitoneally with asbestos had higher levels of proinflammatory M1 macrophages and of TNF-α, IL-1ß, IL-3, IL-10, and IL-12 in the peritoneal lavage, findings linked to asbestos carcinogenesis. Blm+/- mice exposed to asbestos had a significantly shorter survival and higher incidence of mesothelioma compared to controls. We propose that germline BLM+/- mutations increase the susceptibility to asbestos carcinogenesis, enhancing the risk of developing mesothelioma.
Assuntos
Asbestose/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genética , Mesotelioma/genética , RecQ Helicases/genética , Adulto , Idoso , Animais , Asbesto Crocidolita , Família , Feminino , Instabilidade Genômica , Heterozigoto , Humanos , Incidência , Inflamação/patologia , Masculino , Camundongos , Pessoa de Meia-IdadeRESUMO
Precise quantification of copy-number alterations (CNAs) in a tumor genome is difficult. We have applied a comprehensive copy-number analysis method, digital multiplex ligation-dependent probe amplification (digitalMLPA), for targeted gene copy-number analysis in clear cell renal cell carcinoma (ccRCC). Copy-number status of all chromosomal arms and 11 genes was determined in 60 ccRCC samples. Chromosome 3p loss and 5q gain, known as early changes in ccRCC development, as well as losses at 9p and 14q were detected in 56/60 (93.3%), 31/60 (51.7%), 11/60 (18.3%), and 33/60 (55%), respectively. Through gene expression analysis, a significant positive correlation was detected in terms of 14q loss determined using digitalMLPA and downregulation of mRNA expression ratios with HIF1A and L2HGDH (P = .0253 and .0117, respectively). Patients with early metastasis (<1 y) (n = 18) showed CNAs in 6 arms (in median), whereas metastasis-free patients (n = 34) showed those in significantly less arms (3 arms in median) (P = .0289). In particular, biallelic deletion of CDKN2A/2B was associated with multiple CNAs (≥7 arms) in 3 tumors. Together with sequence-level mutations in genes VHL, PBRM1, SETD2, and BAP1, we performed multiple correspondence analysis, which identified the association of 9p loss and 4q loss with early metastasis (both P < .05). This analysis indicated the association of 4p loss and 1p loss with poor survival (both, P < .05). These findings suggest that CNAs have essential roles in aggressiveness of ccRCC. We showed that our approach of measuring CNA through digitalMLPA will facilitate the selection of patients who may develop metastasis.
Assuntos
Carcinoma de Células Renais/genética , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 9/genética , Variações do Número de Cópias de DNA , Neoplasias Renais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/mortalidade , Estudos de Casos e Controles , Deleção Cromossômica , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/mortalidade , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Metástase Neoplásica , Análise de SobrevidaRESUMO
Mesothelioma in situ (MIS) has recently been investigated as a distinct phase of mesothelioma carcinogenesis. The diagnostic criteria proposed for MIS include a loss of BRCA1-associated protein 1 (BAP1) expression detected by immunohistochemistry (IHC). However, the length of time that MIS typically remains an in situ lesion before progression to invasive disease is still unclear. Herein, we report a case of a Japanese woman in her early seventies who had suffered from recurrent pleural effusions for 15 years, during which MIS developed and progressed to invasive mesothelioma. Retrospective diagnosis of partial MIS, fully developed MIS, and invasive disease was made using BAP1 IHC on three biopsy specimens and via clinical observations with radiological images. MIS and invasive lesions revealed BAP1 loss. The interval from partial or full MIS to invasive disease was 14 and 7 years, respectively. These results support a diagnostic strategy combining histomorphology with genomic-based assays including BAP1 IHC in biopsy tissues from a patient with recurrent pleural effusions.
Assuntos
Mesotelioma , Derrame Pleural/complicações , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Biópsia , Diagnóstico Diferencial , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Mesotelioma/diagnóstico , Mesotelioma/patologia , Mesotelioma Maligno/diagnóstico , Mesotelioma Maligno/patologia , Derrame Pleural/patologia , Neoplasias Pleurais/diagnóstico , Neoplasias Pleurais/patologia , Prognóstico , Estudos RetrospectivosRESUMO
We used a custom-made comparative genomic hybridization array (aCGH; average probe interval 254 bp) to screen 33 malignant mesothelioma (MM) biopsies for somatic copy number loss throughout the 3p21 region (10.7 Mb) that harbors 251 genes, including BRCA1 (breast cancer 1)-associated protein 1 (BAP1), the most commonly mutated gene in MM. We identified frequent minute biallelic deletions (<3 kb) in 46 of 251 genes: four were cancer-associated genes: SETD2 (SET domain-containing protein 2) (7 of 33), BAP1 (8 of 33), PBRM1 (polybromo 1) (3 of 33), and SMARCC1 (switch/sucrose nonfermentable- SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily c, member 1) (2 of 33). These four genes were further investigated by targeted next-generation sequencing (tNGS), which revealed sequence-level mutations causing biallelic inactivation. Combined high-density aCGH and tNGS revealed biallelic gene inactivation in SETD2 (9 of 33, 27%), BAP1 (16 of 33, 48%), PBRM1 (5 of 33, 15%), and SMARCC1 (2 of 33, 6%). The incidence of genetic alterations detected is much higher than reported in the literature because minute deletions are not detected by NGS or commercial aCGH. Many of these minute deletions were not contiguous, but rather alternated with segments showing oscillating copy number changes along the 3p21 region. In summary, we found that in MM: (i) multiple minute simultaneous biallelic deletions are frequent in chromosome 3p21, where they occur as distinct events involving multiple genes; (ii) in addition to BAP1, mutations of SETD2, PBRM1, and SMARCC1 are frequent in MM; and (iii) our results suggest that high-density aCGH combined with tNGS provides a more precise estimate of the frequency and types of genes inactivated in human cancer than approaches based exclusively on NGS strategy.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 3/genética , Hibridização Genômica Comparativa , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias Pulmonares/genética , Mesotelioma/genética , Alelos , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genoma Humano , Humanos , Mesotelioma Maligno , Família Multigênica , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos TestesRESUMO
We detected low levels of acetylation for histone H3 tail lysines in malignant mesothelioma (MM) cell lines resistant to histone deacetylase inhibitors. To identify the possible genetic causes related to the low histone acetylation levels, whole-exome sequencing was conducted with MM cell lines established from eight patients. A mono-allelic variant of BRD1 was common to two MM cell lines with very low acetylation levels. We identified 318 homozygous protein-damaging variants/mutations (18-78 variants/mutations per patient); annotation analysis showed enrichment of the molecules associated with mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complexes and co-activators that facilitate initiation of transcription. In seven of the patients, we detected a combination of variants in histone modifiers or transcription factors/co-factors, in addition to variants in mSWI/SNF. Direct sequencing showed that homozygous mutations in SMARCA4, PBRM1 and ARID2 were somatic. In one patient, homozygous germline variants were observed for SMARCC1 and SETD2 in chr3p22.1-3p14.2. These exhibited extended germline homozygosity and were in regions containing somatic mutations, leading to a loss of BAP1 and PBRM1 expression in MM cell line. Most protein-damaging variants were heterozygous in normal tissues. Heterozygous germline variants were often converted into hemizygous variants by mono-allelic deletion, and were rarely homozygous because of acquired uniparental disomy. Our findings imply that MM might develop through the somatic inactivation of mSWI/SNF complex subunits and/or histone modifiers, including BAP1, in subjects that have rare germline variants of these transcription regulators and/or transcription factors/co-factors, and in regions prone to mono-allelic deletion during oncogenesis.
Assuntos
Proteínas Cromossômicas não Histona/genética , Mutação em Linhagem Germinativa , Neoplasias Pulmonares/genética , Mesotelioma/genética , Fatores de Transcrição/genética , Acetilação , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Histona Acetiltransferases , Chaperonas de Histonas , Histonas/metabolismo , Humanos , Mesotelioma Maligno , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genéticaRESUMO
Malignant mesothelioma (MM) is an asbestos-related malignancy arising from surface serosal cells of pleural and peritoneal cavities. Somatic mutations of BRCA1 associated protein-1 (BAP1) gene were recently found in MM as well as in uveal melanoma and kidney cancer among the Caucasian and Japanese people. However, frequency of mutations varies among the reported studies, which might be due to presence of undetected gross rearrangements of BAP1 gene that might escape detection by sequencing strategy. We investigated the presence and frequency of gross genomic rearrangements in the BAP1 gene by multiplex ligation-dependent probe amplification (MLPA) in 17 Japanese cases of MM tumors. We found five tumors with partial deletion of BAP1 gene; each tumors displayed partial deletion of exons 1-4 (MM39), exons 1-5 (MM48), exons 11-17 (MM57), exons 1-15 (MM19) and exons 1-16 (MM21). Two tumors (MM34, MM14) had biallelic deletion and four tumors (MM29, MM35, MM45 and MM56) had monoallelic deletion of entire BAP1 gene. Therefore, MLPA analysis revealed large gene rearrangements of BAP1 gene in 65% of MM (11/17). Unusually high frequency of large deletions indicates that the 3p21 chromosomal region surrounding BAP1 gene is structurally unstable. MLPA was useful in characterizing both monoallelic and biallelic deletion of BAP1 gene precisely at exon level.
Assuntos
Sequência de Bases , Instabilidade Cromossômica , Cromossomos Humanos Par 3/genética , Éxons , Neoplasias Pulmonares/genética , Mesotelioma/genética , Deleção de Sequência , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Povo Asiático , Feminino , Humanos , Japão , Masculino , Mesotelioma MalignoRESUMO
We cultured human hair follicle-derived keratinocytes (FDKs) from plucked hairs. To gain insight into gene expression signatures that can distinguish atopic dermatitis from non-atopic controls without skin biopsies, we undertook a comparative study of gene expression in FDKs from adult donors with atopic dermatitis and non-atopic donors. FDK primary cultures (atopic dermatitis, n = 11; non-atopic controls, n = 7) before and after interferon gamma (IFN-γ) treatment were used for microarray analysis and quantitative RT-PCR. Comparison of FDKs from atopic and non-atopic donors indicated that the former showed activated pathways with innate immunity and decreased pathways of cell growth, as indicated by increased NLRP2 expression and decreased DKK1 expression, respectively. Treatment with IFN-γ induced the enhanced expression of IL32, IL1B, IL8, and CXCL1 in the cells from atopic donors compared to that in cells from non-atopic donors at 24 h after treatment. IL1B expression in FDKs after IFN-γ treatment correlated with IL32 expression. We hypothesized that overexpression of IL32 in hair follicle keratinocytes of patients with atopic dermatitis would lead to the excessive production of pro-IL1ßï and that the activation of IL1ß from pro-IL1ß by inflammasome complex, in which NLRP2 protein might be involved, would be augmented. This is the first report to show enhanced induction of cytokine/chemokine genes by IFN-γ in atopic dermatitis using cultured FDKs.
RESUMO
Few studies have examined the influence of pre-exercise meals with different glycaemic indices (GIs) on substrate oxidation and non-homeostatic appetite (i.e. food reward) in adults of various ages and ethnicities. We aimed to examine the effects of pre-exercise high and low GI meals on substrate oxidation and food reward in middle-aged Japanese women. This randomised crossover trial included fifteen middle-aged women (aged 40â 9 ± 6â 5 years, mean ± sd). The participants consumed a high or low GI breakfast at 09.00 and rested until 11.00. Thereafter, participants performed a 60-min walk at 50 % of their estimated maximum oxygen uptake (11.00-12.00) and rested until 13.00. Expired gas samples were collected every 30 min prior to walking, and samples were collected continuously throughout the walking and post-walking periods. Blood samples and subjective appetite ratings were collected every 30 min, except during walking. The Leeds Food Preference Questionnaire in Japanese (LFPQ-J) was used to assess food reward at 09.00, 10.00, and 13.00 h. The cumulative fat oxidation during exercise was higher in the low GI trial than in the high GI trial (P = 0â 03). The cumulative carbohydrate oxidation during walking was lower in the low GI trial than in the high GI trial (P = 0â 01). Trial-by-time interactions were not found for any food-reward parameters between trials. Low GI meals elicited enhanced fat oxidation during a subsequent 60-min walk in middle-aged women. However, meals with different GIs did not affect food reward evaluated over time in the present study.
Assuntos
Apetite , Índice Glicêmico , Adulto , Pessoa de Meia-Idade , Humanos , Feminino , Glicemia/metabolismo , Consumo de Oxigênio , Carboidratos da Dieta/metabolismo , Oxigênio , RefeiçõesRESUMO
In the present study, we analyzed genomic alterations of BRCA1-associated protein 1 (BAP1) in 23 malignant mesotheliomas (MMs), 16 epithelioid and seven non-epithelioid, consisting of 18 clinical specimens and five established cell lines. In examining these samples for homozygous deletions and sequence-level mutations, we found biallelic BAP1 gene alterations in 14 of 23 MMs (61%). Seven of these 14 MMs had homozygous deletions of the partial or entire BAP1 gene, another five had sequence-level mutations, including small deletions, a nonsense mutation, and missense mutations with additional monoallelic deletions, and the remaining two had homozygous mutations without allelic loss. All but one of the 14 BAP1 gene mutations were found in the epithelioid-type MMs; BAP1 mutations were found in 13 of 16 epithelioid-type MMs, but in only one of seven non-epithelioid-type MMs (13/16 vs 1/7; P = 0.005). There was no BAP1 mRNA expression in MMs with biallelic deletion and repressed expression was confirmed in MM specimens with deletion/mutation as compared with Met5a, SV40-transformed normal mesothelial cells. Western blot showed that seven of eight epithelioid MMs analyzed were BAP1 negative. Immunostaining with anti-BAP1 antibody in normal lung tissues revealed clear nuclear staining of normal mesothelial cells. No nuclear staining was observed among BAP1 mutation-positive MM tumors, whereas nuclear staining was observed among BAP1 mutation-negative MM tumors. These results suggest that the lack of the tumor suppressor BAP1 may be more specifically involved in the pathogenesis of epithelioid MM rather than non-epithelioid MM, and would be useful for diagnosis of epithelioid-type MM.
Assuntos
Deleção de Genes , Mesotelioma/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Linhagem Celular Tumoral , Humanos , Mutação , Neoplasias Epiteliais e Glandulares/genéticaRESUMO
BACKGROUND: Malignant mesothelioma is an aggressive tumor of serosal surfaces that is closely associated with asbestos exposure which induces oxidative stress. Heme oxygenase (HO)-1, a rate-limiting enzyme of heme degradation, plays a protective role against oxidative stress. The HO-1 gene promoter carries (GT)n repeats whose number is inversely related to transcriptional activity of the HO-1 gene. METHODS: To investigate the relationship between the length polymorphism of (GT)n repeats and mesothelioma susceptibility, we analyzed the HO-1 promoter in 44 asbestos-exposed subjects without mesothelioma and 78 asbestos-exposed subjects with mesothelioma using PCR-based genotyping. RESULTS: The number of repeats ranged from 16 to 38, with two peaks at 23 and 30 repeats. Polymorphisms of (GT)n repeats were grouped into two classes of alleles, short (S) (<24) and long (L) (≥24), and three genotypes: L/L, L/S, and S/S. The proportions of allele frequencies in class L as well as genotypic frequencies of L allele carriers (L/L and L/S) were significantly higher in the asbestos-exposed subjects with mesothelioma than in those without mesothelioma. CONCLUSION: The findings of this study suggest that long (GT)n repeats in the HO-1 gene promoter are associated with a higher risk of malignant mesothelioma in the Japanese population.
Assuntos
Predisposição Genética para Doença , Heme Oxigenase-1/genética , Mesotelioma/genética , Neoplasias Pleurais/genética , Polimorfismo Genético , Regiões Promotoras Genéticas , Idoso , Idoso de 80 Anos ou mais , Alelos , Amianto/efeitos adversos , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Mesotelioma/etiologia , Pessoa de Meia-Idade , Neoplasias Pleurais/etiologia , Fatores de RiscoRESUMO
The most common malignancies that develop in carriers of BAP1 germline mutations include diffuse malignant mesothelioma, uveal and cutaneous melanoma, renal cell carcinoma, and less frequently, breast cancer, several types of skin carcinomas, and other tumor types. Mesotheliomas in these patients are significantly less aggressive, and patients require a multidisciplinary approach that involves genetic counseling, medical genetics, pathology, surgical, medical, and radiation oncology expertise. Some BAP1 carriers have asymptomatic mesothelioma that can be followed by close clinical observation without apparent adverse outcomes: they may survive many years without therapy. Others may grow aggressively but very often respond to therapy. Detecting BAP1 germline mutations has, therefore, substantial medical, social, and economic impact. Close monitoring of these patients and their relatives is expected to result in prolonged life expectancy, improved quality of life, and being cost-effective. The co-authors of this paper are those who have published the vast majority of cases of mesothelioma occurring in patients carrying inactivating germline BAP1 mutations and who have studied the families affected by the BAP1 cancer syndrome for many years. This paper reports our experience. It is intended to be a source of information for all physicians who care for patients carrying germline BAP1 mutations. We discuss the clinical presentation, diagnostic and treatment challenges, and our recommendations of how to best care for these patients and their family members, including the potential economic and psychosocial impact.
Assuntos
Neoplasias Pulmonares , Melanoma , Mesotelioma Maligno , Mesotelioma , Neoplasias Cutâneas , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirurgia , Melanoma/genética , Mesotelioma/diagnóstico , Mesotelioma/genética , Mesotelioma/cirurgia , Qualidade de Vida , Neoplasias Cutâneas/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
Malignant pleural mesothelioma is a refractory tumor with increasing incidence. In the present study, we established six mesothelioma cell lines possessing two allele deletions of the p16(INK4A) gene and one allele deletion of the neurofibromatosis type 2 gene, MM16, MM21, MM26, MM35, MM46 and MM56, from pleural effusion fluids or surgically resected tumors of Japanese patients. MM21, MM26 and MM46 cells failed to develop tumors in BALB/c-nude mice following subcutaneous inoculation. MM16 and MM35 cells slowly generated tumors at the site of subcutaneous inoculation in BALB/c-nude mice, but lost the expression of mesothelioma-related markers such as calretinin, D2-40 and Wilms' tumor 1 in the subcutaneous tumors. On the other hand, MM56 cells rapidly generated tumors with the expression of calretinin and D2-40 in BALB/c-nude mice following subcutaneous inoculation. In addition, orthotopic implantation of MM56 cells into BALB/c-nude mice developed diffusely growing thoracic tumors by 3 weeks after implantation. Pleural effusions were observed in these mice 4 weeks after implantation. Thoracic tumors invaded aggressively into the chest wall 5 weeks after implantation and often metastasized into the lung, rib, peritoneum and pericardial cavity. On the pleural surface, MM56 cells were growing as single or multiple cell layers with the reactive mesothelium of recipient mice. These results indicate that MM56 cells can behave in a manner characteristic of human malignant pleural mesothelioma in the thoracic cavity of BALB/c-nude mice. The in vivo model using MM56 cells may be useful for studying the biological behavior of malignant pleural mesothelioma and developing its diagnostic and therapeutic strategies.
Assuntos
Mesotelioma/patologia , Neoplasias Pleurais/patologia , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Genes da Neurofibromatose 2 , Genes p16 , Humanos , Imuno-Histoquímica , Masculino , Mesotelioma/química , Mesotelioma/genética , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Transplante de Neoplasias , Neoplasias Pleurais/química , Neoplasias Pleurais/genética , Transplante HeterólogoRESUMO
In the epidermis, keratinocytes are involved in physical and first-line immune protection of the host. In this study, we analyzed the molecular responses to certain contact sensitizers (2,4-dinitrochlorobenzene and NiSO(4)) and irritants (sodium dodecyl sulfate and benzalkonium chloride) in cultured human keratinocytes from the bulge region of a plucked hair follicle (bulge-derived keratinocytes [BDKs]) and compared these molecular responses to those with the human monocytic leukemia cell line, THP-1. The BDKs, individually established without invasive biopsies, showed high reactivity to these stimulants. As a primary response to the contact sensitizers, the NRF2-mediated signaling pathway was upregulated in BDKs and THP-1. The expression of IL1B and IL8 genes was not induced by the irritants but by the sensitizers in THP-1. However, the expression of the IL1B and IL8 genes was induced at higher levels by the irritants in BDKs than by the sensitizers. Many genes orchestrating keratinocyte differentiation, including ID2, were significantly upregulated in response to the sensitizers in BDKs but not those in THP-1. The use of the ID2 gene to discriminate between sensitizers and irritants might be effective as a novel marker for application during in vitro sensitization with BDKs.
Assuntos
Diferenciação Celular , Folículo Piloso/citologia , Proteína 2 Inibidora de Diferenciação/metabolismo , Irritantes/toxicidade , Queratinócitos/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Adulto , Apoptose/efeitos dos fármacos , Biomarcadores , Linhagem Celular Tumoral , Células Cultivadas , Dermatite de Contato/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteína 2 Inibidora de Diferenciação/genética , Interleucinas/genética , Interleucinas/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
Malignant mesothelioma is associated with the exposure to asbestos fibers. Recent discovery of the BAP1 cancer syndrome, a Mendelian disorder with high-penetrance autosomal dominant inheritance fostered the genotyping for nucleotide-level or larger structural alteration of germline DNA. Inherited heterozygous mutations of the BAP1 gene increase the susceptibility to carcinogenic fibers, leading to a concept of gene x environment interaction (GxE) as a pathogenetic mechanism of mesothelioma. Several studies on cohorts of unselected patients with mesothelioma or on familial/early-onset cohorts of mesothelioma cases converged on BAP1 as the more frequent germline mutated gene, followed by other genes involved in DNA repair and homologous recombination. Evidence has been emerging that patients with mesothelioma carrying germline mutations of BAP1 and of other genes, such as those involved in DNA repair and tumor suppressor genes, have better prognosis and higher chemosensitivity when compared with patients with germline wildtype Bap1. We report here a germline genomic analysis targeted 22 genes in a cohort of 101 Japanese patients irrespective of asbestos exposure, age at diagnosis, or personal or family history of cancer. By comparing the results with the Human Genetic Variation Database (HGVD) and the Genome Aggregation Database (gnomAD) we selected rare germline variants with a Combined Annotation Dependent Depletion (CADD) >20. We show here that 31 of 101 subjects were carrying 25 rare variants in 14 genes, neither reported in the HGVD nor in the gnomAD database for 14/25 variants. Besides pathogenic variants of BAP1, rare missense variants were found in genes encoding lysine-specific histone methyltransferase SETD2 and SETDB1 and genes encoding subunits of the mSWI/SNF chromatin remodeling complex. The complete scenario of the genetic background consisting of pathogenic germline variants required for the predisposition and GxE for pathogenesis of mesothelioma appears complex, and further large-scale studies are warranted.
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Malignant pleural mesothelioma (MPM) is strongly associated with occupational or environmental asbestos exposure and arises from neoplastic transformation of mesothelial cells in the pleural cavity. The only standard initial treatment for unresectable MPM is combination chemotherapy with cisplatin (CDDP) and pemetrexed (PEM). Further, CDDP/PEM is the only approved regimen with evidence of prolonged overall survival (OS), although the median OS for patients treated with this regimen is only 12 months after diagnosis. Thus, the development of new therapeutic strategies has been investigated for approximately 20 years. In contrast to recent advances in personalized lung cancer therapies, diagnostic and prognostic biomarker research has just started in mesothelioma. Epigenetic alterations include DNA methylation, histone modifications, and other chromatin-remodeling events. These processes are involved in numerous cellular processes including differentiation, development, and tumorigenesis. Epigenetic modifications play an important role in gene expression and regulation related to malignant MPM phenotypes and histological subtypes. An immune checkpoint PD-1 inhibitor, nivolumab, was approved as second-line therapy for patients who had failed initial chemotherapy, based on the results of the MERIT study. Various clinical immunotherapy trials are ongoing in patients with advanced MPM. In this review, we describe recent knowledge on epigenetic alterations, which might identify candidate therapeutic targets and immunotherapeutic regimens under development for MPM.
RESUMO
Basal cell nevus syndrome (BCNS or Gorlin syndrome, OMIM: 109400) is a rare autosomal dominant disorder with high penetrance. It is characterized by developmental anomalies and predisposition to tumors (for example, basal cell carcinoma (BCC) and medulloblastoma). PTCH1, the human homolog of the Drosophila patched gene, was identified as a gene responsible for BCNS. The PTCH1 protein is a Hedgehog (Hh) protein receptor and is pivotal for early development, stem cell maintenance and/or differentiation. We analyzed the six Japanese families with BCNS and identified six germline mutations in the PTCH1 gene. One family had a nonsense mutation (c.1196G>A), one had a 1-bp deletion (c.2029delA), two had 2-bp deletions (c.239_240delGA and c.1670_1671delCA) and one had a 58-bp duplication (c.1138_1195dup). They caused premature termination, resulting in the truncation of the PTCH1 protein. Analysis of a high-density single nucleotide polymorphism (SNP) mapping array showed a large approximately 1.2-Mb deletion, including the PTCH1 gene in one allele, in a family in which PTCH1 mutations were not identified at the sequence level. These data indicated that all the six families who were diagnosed with BCNS had mutations in the PTCH1 gene and that a single copy of a PTCH1 mutation causes BCNS.
Assuntos
Síndrome do Nevo Basocelular/genética , Mutação em Linhagem Germinativa/genética , Receptores de Superfície Celular/genética , Adulto , Povo Asiático/genética , Sequência de Bases , Criança , Pré-Escolar , Pontos de Quebra do Cromossomo , Feminino , Humanos , Japão , Masculino , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Patched , Receptor Patched-1 , Linhagem , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Human keratinocyte strains derived from the bulge region of plucked human follicles were successfully established from all 43 donors (age 24-76) regardless of the age and gender. The total cell number, number of population doublings and population doubling time were similar among the strains. These bulge-derived keratinocytes, BDKs, expressed keratin family genes specific to basal cell layers of the epidermis. They also expressed CD34, one of the bulge stem cell marker genes. The growth behavior and positivity of CD34 indicate that BDKs contain stem cells. BDKs were cultured until confluency or treated with CaCl2 to induce differentiation. Morphology and expression of keratin family genes in BDKs before and after differentiation induction with CaCl2 were similar to those of epidermal keratinocytes obtained from skin biopsies (NHEKs). However, expression levels of keratin-10, a prickle cell layer marker, in CaCl2-treated BDKs were lower than those in CaCl2-treated NHEKs. Higher expression of integrin-alpha6, a basal cell layer marker, was also noted in BDKs than in NHEKs after differentiation induction. Expression of stem cell marker genes other than CD34, including CD200, Sox2 and NANOG, was about the same at confluency in both cells, but significantly higher in BDKs than NHEKs after differentiation. These results indicate that BDKs were more refractory to differentiation than NHEKs. We then examined Wnt signaling inhibitor genes, DKK-3 and WIF-1 that function as tumor suppressors. DKK-3 expression decreased in both BDKs and NHEKs after CaCl2-induced differentiation. Expression of WIF-1 decreased 50% in BDKs one day after CaCl2 treatment and remained low, but was induced 1.7 times in NHEKs one day after CaCl2 treatment and further induced thereafter (>2.5 times), suggesting that WIF-1 may be involved in maintaining the differentiation-refractory status of BDKs. Since cancer stem cells in the skin have been reported to be similar to bulge-derived stem cells, our BDK strains may be of use in studying characteristics of cancer stem cells of the epidermis.
Assuntos
Diferenciação Celular/fisiologia , Folículo Piloso/citologia , Queratinócitos/fisiologia , Adulto , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/fisiologia , Idoso , Biópsia , Cálcio/farmacologia , Técnicas de Cultura de Células/métodos , Proliferação de Células , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Perfilação da Expressão Gênica , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Pele/patologia , Adulto JovemRESUMO
PURPOSE: We hypothesized that four criteria could help identify malignant mesotheliomas (MMs) most likely linked to germline mutations of BAP1 or of other genes: family history of MM, BAP1-associated cancers, or multiple malignancies; or age younger than 50 years. PATIENTS AND METHODS: Over the course of 7 years, 79 patients with MM met the four criteria; 22 of the 79 (28%) reported possible asbestos exposure. They were screened for germline BAP1 mutations by Sanger sequencing and by targeted next-generation sequencing (tNGS) for germline mutations in 55 additional cancer-linked genes. Deleterious mutations detected by tNGS were validated by Sanger sequencing. RESULTS: Of the 79 patients, 43 (16 probands and 27 relatives) had deleterious germline BAP1 mutations. The median age at diagnosis was 54 years and median survival was 5 years. Among the remaining 36 patients with no BAP1 mutation, median age at diagnosis was 45 years, median survival was 9 years, and 12 had deleterious mutations of additional genes linked to cancer. When compared with patients with MMs in the SEER cohort, median age at diagnosis (72 years), median survival for all MM stages (8 months), and stage I (11 months) were significantly different from the 79 patients with MM in the current study ( P < .0001). CONCLUSION: We provide criteria that help identify a subset of patients with MM who had significantly improved survival. Most of these patients were not aware of asbestos exposure and carried either pathogenic germline mutations of BAP1 or of additional genes linked to cancer, some of which may have targeted-therapy options. These patients and their relatives are susceptible to development of additional cancers; therefore, genetic counseling and cancer screening should be considered.