RESUMO
The systematic identification of genetic events driving cellular transformation and tumor progression in the absence of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified both known tumor suppressor genes and novel oncogenic drivers of cuSCC. Functional analysis confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely heterogeneous genetic landscape of cuSCC initiation and progression, which can be harnessed to better understand skin oncogenic etiology and prioritize therapeutic candidates.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Transformação Celular Neoplásica/genética , Queratinócitos/patologia , Mutagênese Insercional/métodos , Análise de Sequência de DNA/métodos , Neoplasias Cutâneas/genética , Proteína de Ligação a CREB/genética , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/patologia , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Coativador 2 de Receptor Nuclear/genética , Neoplasias Cutâneas/patologiaRESUMO
Entamoeba histolytica causes amoebic liver abscess (ALA) in humans. The injury of target cells by E. histolytica includes processes controlled by the ubiquitin Ehub. Previously, we found immunodominance of Ehub glycan moieties using immunized rabbits. In this work, we analysed dominance of antibodies to the glycoprotein Ehub in the sera from 52 patients with ALA. Controls were sera from 20 healthy people living in endemic areas with a high seroprevalence of antibodies to amoebas, and 20 patients with alcoholic hepatitis (AH) to rule out the cross-reaction of Ehub with autoantibodies induced by liver damage. Antigens were trophozoite extract, glycoprotein Ehub and the recombinant protein E. histolytica recombinant ubiquitin (rEhub). The sera from healthy volunteers and patients with AH do not have antibodies to glycoprotein Ehub. Surprisingly, only the antibodies from patients with ALA recognized the glycoprotein Ehub, and some sera gave a faint reaction with the recombinant protein, especially because evolutionarily, the ubiquitin is conserved between species. This is the first report demonstrating that antibodies to ubiquitin Ehub are induced exclusively in patients with invasive amoebiasis, and the antibody response is mainly to the glycoprotein, indicating glycans are immunodominant. Inhibitors of the Ehub glycans could be potential treatment for amoebiasis by selectively damaging trophozoites.
Assuntos
Amebíase , Disenteria Amebiana , Entamoeba histolytica , Amebíase/tratamento farmacológico , Animais , Anticorpos Antiprotozoários , Formação de Anticorpos , Humanos , Coelhos , Proteínas Recombinantes , Estudos Soroepidemiológicos , Trofozoítos , UbiquitinaRESUMO
The Mu-opioid receptor (MOR) has been implicated in tumorigenesis and metastasis. Methylnaltrexone (MNTX), an antagonist of MOR, has shown to inhibit tumor growth and metastasis in lung cancer cell lines. The effect of MNTX on other cell lines such as head and neck squamous cell carcinoma (HNSCC) has not been investigated. We measured the expression and activity of the receptor in different HNSCC cell lines. Then, we evaluated the impact of modulating the expression MOR and the effect of MNTX on the proliferation, clonogenic activity, invasion, and migration of two HNSCC (FaDu and MDA686Tu) cell lines expressing MOR and one cell line (UMSCC47) not expressing the receptor. We also evaluated the impact of MNTX on tumor growth and metastasis formation in vivo. Activation of the receptor with [d-Ala2,N-Me-Phe4, Gly5-ol] (DAMGO) caused a significant reduction in cyclic adenosine monophosphate levels in FaDu cells. Knockdown of MOR inhibited in vitro aggressive cell behaviors on FaDu and MDA686Tu cells and correlated with a reduction in markers of epithelial-mesenchymal transition. In vitro studies showed that MNTX strongly inhibited the proliferation, clonogenic activity, invasion, and migration of FaDu and MDA686Tu cells but has no effect on UMSCC47 cells. In vivo experiments demonstrated that MNTX suppresses tumor growth in HNSCC cell tumor-bearing mice. Our studies indicate that MOR could be considered as a therapeutic target to treat HNSCC.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Naltrexona/análogos & derivados , Antagonistas de Entorpecentes/farmacologia , Receptores Opioides mu/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Nus , Naltrexona/farmacologia , Invasividade Neoplásica , Compostos de Amônio Quaternário/farmacologia , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The ubiquitin-proteasome system plays a central role performing several functions to maintain parasite homeostasis. We have reported the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin (EhUb). Here we examined the immunogenicity and antigenicity of carbohydrates in EhUbiquitin. Rabbits were immunized with purified EhUbiquitin or purified recombinant rUb expressed by E. coli. Using Western Blot, we explored the immunogenicity and antigenicity of protein portion and carbohydrates moiety. Interestingly, immunized rabbits produced antibodies to both Ub glycoprotein and rUb; but antibodies against carbohydrates were immunodominant, rather than antibodies to the protein moiety of EhUbiquitin. In addition, we observed that antibodies to protein moiety are not conserved in serum unless antigen is continually administrated. Conversely, anti-Ub glycoprotein antibodies are well maintained in circulation. In humans, infection with Entamoeba histolytica induces strong IgG anti-Ub response. The human antibodies recognize both, the protein moieties and the glycosylated structure. Entamoeba histolytica ubiquitin is immunogenic and antigenic. The glycan moieties are immunodominant and induces IgG. These data open the door to use carbohydrates as potential targets for diagnose tests, drugs and vaccine to prevent this parasitic disease.
Assuntos
Entamoeba histolytica/imunologia , Entamebíase/prevenção & controle , Epitopos Imunodominantes , Polissacarídeos/imunologia , Ubiquitina/imunologia , Animais , Anticorpos Antiprotozoários/análise , Anticorpos Antiprotozoários/biossíntese , Western Blotting , Entamebíase/imunologia , Glicosilação , Humanos , CoelhosRESUMO
Epithelial ovarian cancer (EOC) is a deadly cancer, and its prognosis has not been changed significantly during several decades. To seek new therapeutic targets for EOC, we performed an in vivo dropout screen in human tumor xenografts using a pooled shRNA library targeting thousands of druggable genes. Then, in follow-up studies, we performed a second screen using a genome-wide CRISPR/Cas9 library. These screens identified 10 high-confidence drug targets that included well-known oncogenes such as ERBB2 and RAF1, and novel oncogenes, notably KPNB1, which we investigated further. Genetic and pharmacological inhibition showed that KPNB1 exerts its antitumor effects through multiphase cell cycle arrest and apoptosis induction. Mechanistically, proteomic studies revealed that KPNB1 acts as a master regulator of cell cycle-related proteins, including p21, p27, and APC/C. Clinically, EOC patients with higher expression levels of KPNB1 showed earlier recurrence and worse prognosis than those with lower expression levels of KPNB1. Interestingly, ivermectin, a Food and Drug Administration-approved antiparasitic drug, showed KPNB1-dependent antitumor effects on EOC, serving as an alternative therapeutic toward EOC patients through drug repositioning. Last, we found that the combination of ivermectin and paclitaxel produces a stronger antitumor effect on EOC both in vitro and in vivo than either drug alone. Our studies have thus identified a combinatorial therapy for EOC, in addition to a plethora of potential drug targets.
Assuntos
Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , beta Carioferinas/genética , beta Carioferinas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Epitelial do Ovário , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Ivermectina/farmacologia , Mutação com Perda de Função/genética , Neoplasias Epiteliais e Glandulares/metabolismo , Oncogenes , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , RNA Interferente Pequeno/genéticaRESUMO
Entamoeba histolytica harbors an extensive intracellular distribution of ubiquitin-proteasome systems important for numerous cellular processes. However, glycosylation studies of ubiquitin-proteasome components have not yet been elucidated. Here we report the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE), Nanoelectrospray Ionization-Tandem Mass Spectrometry (NSI-MS), Matrix-Assisted Laser-Desorption time-of-flight Mass Spectrometry (MALDI-TOF MS) and Gas Chromatography-Mass Spectrometry (GC-MS) analysis. To our knowledge, the data presented in this report represents the first structural glycomics analysis of E. histolytica ubiquitin, while most of the reports are performed on whole parasitic glycan profiles. The glycan profile of E. histolytica ubiquitin has high mannose N-glycan structures. The N-linked glycan profile showed fragments from Hex3HexNAc2 to Hex9HexNAc2. Based in our findings and ubiquitin function, we hypothesize that the same ubiquitin Asn-Asp-Ser sequon carries heterogenic glycosylations, at different metabolic pathway stages according to ubiquitin functional requirements. Finally, we propose a set of possible high mannose N-glycan structures that will help to elucidate the ubiquitin biochemical composition and may well represent good targets for anti-amoebic drugs.
Assuntos
Entamoeba histolytica/química , Polissacarídeos/química , Ubiquitina/metabolismo , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Glicosilação , Espectrometria de Massas/métodos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Reação do Ácido Periódico de Schiff , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trofozoítos/química , Ubiquitina/genética , Ubiquitina/imunologia , Ubiquitina/isolamento & purificaçãoRESUMO
Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets.
Assuntos
Carcinoma Hepatocelular , Elementos de DNA Transponíveis , Transição Epitelial-Mesenquimal/genética , Neoplasias Hepáticas , Mutagênese , Proteínas de Neoplasias , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Movimento Celular/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Nus , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proto-Oncogene MasRESUMO
Triple-negative breast cancer (TNBC) has the worst prognosis of any breast cancer subtype. To better understand the genetic forces driving TNBC, we performed a transposon mutagenesis screen in a phosphatase and tensin homolog (Pten) mutant mice and identified 12 candidate trunk drivers and a much larger number of progression genes. Validation studies identified eight TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1 Down-regulation of TRPS1 in TNBC cells promoted epithelial-to-mesenchymal transition (EMT) by deregulating multiple EMT pathway genes, in addition to increasing the expression of SERPINE1 and SERPINB2 and the subsequent migration, invasion, and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC and discovered genes with potential clinical importance in TNBC.
Assuntos
Adenocarcinoma/genética , Elementos de DNA Transponíveis , Neoplasias Mamárias Experimentais/genética , PTEN Fosfo-Hidrolase/genética , Adenocarcinoma/secundário , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Genes Supressores de Tumor , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares , Neoplasias Mamárias Experimentais/patologia , Camundongos Transgênicos , Mutagênese , Mutação de Sentido Incorreto , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Repressoras , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
BACKGROUND & AIMS: High-throughput sequencing technologies have identified thousands of infrequently mutated genes in hepatocellular carcinomas (HCCs). However, high intratumor and intertumor heterogeneity, combined with large numbers of passenger mutations, have made it difficult to identify driver mutations that contribute to the development of HCC. We combined transposon mutagenesis with a high-throughput screen of a small-hairpin RNA (shRNA) library to identify genes and pathways that contribute to HCC development. METHODS: Sleeping beauty transposons were mobilized in livers of transgenic mice predisposed to develop hepatocellular adenoma and HCC owing to expression of the hepatitis B virus surface antigen. This whole-genome mutagenesis technique was used to generate an unbiased catalogue of candidate cancer genes (CCGs). Pooled shRNA libraries targeting 250 selected CCGs then were introduced into immortalized mouse liver cells and the cells were monitored for their tumor-forming ability after injection into nude mice. RESULTS: Transposon-mediated mutagenesis identified 1917 high-confident CCGs and highlighted the importance of Ras signaling in the development of HCC. Subsequent pooled shRNA library screening of 250 selected CCGs validated 27 HCC tumor-suppressor genes. Individual shRNA knockdown of 4 of these genes (Acaa2, Hbs1l, Ralgapa2, and Ubr2) increased the proliferation of multiple human HCC cell lines in culture and accelerated the formation of xenograft tumors in nude mice. The ability of Ralgapa2 to promote HCC cell proliferation and tumor formation required its inhibition of Rala and Ralb. Dual inhibition of Ras signaling via Ral and Raf, using a combination of small-molecule inhibitor RBC8 and sorafenib, reduced the proliferation of HCC cells in culture and completely inhibited their growth as xenograft tumors in nude mice. CONCLUSIONS: In a 2-step forward genetic screen in mice, we identified members of the Ral guanosine triphosphatase-activating protein pathway and other proteins as suppressors of HCC cell proliferation and tumor growth. These proteins might serve as therapeutic targets for liver cancer.
Assuntos
Carcinoma Hepatocelular/genética , Proteínas Ativadoras de GTPase/fisiologia , Genes Supressores de Tumor , Neoplasias Hepáticas Experimentais/genética , Proteínas ral de Ligação ao GTP/fisiologia , Animais , Proliferação de Células/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Transdução de Sinais/genéticaRESUMO
Amoebiasis is the third cause of death due to parasites in the world. Although, numerous serodiagnostic and salivary tests have been developed, the majority of these assays lack sensitivity in endemic zones to detect acute amoebic liver abscess. The two main limiting factors to develop reliable assays are the high levels of anti-amoeba antibodies in populations living in endemic zones, and the proteolysis of amoebic extracts even treated with inhibitors. Our group reported a method to preserve amoebic antigens without using enzymatic inhibitors (IC:MC fraction) that shows stability for years. Here we describe the development of a serologic ELISA to diagnose amoebiasis made with IC: MC antigens, and its validation for clinical use in endemic areas. In our study, we included sera from 66 patients diagnosed with acute amoebic liver abscess and 33 volunteers living in an endemic area for amoebiasis. Our assay was compared with an indirect haemagglutination assay (IHA) an ELISA elaborated with antigens derived from untreated trophozoites. The ELISA made with IC: MC antigens presented more reproducibility compared to other assays. Sera from 95% ALA patients showed a positive value. The ELISA (IC: MC) detected 97% of patients with ALA compared to an 81% using IHA. The parameters of ELISA (vs. IHA) were Sensitivity 98% (81%), Specificity 96% (97%), Positive predictive value 98% (96%), Negative predictive value 96% (73%) and Accuracy 98% (87%). A negative serologic test does not rule out the diagnosis of invasive amoebiasis. The ELISA made with antigens preserved without using enzymatic inhibitors has valuable serodiagnostic value to diagnose acute amoebic liver abscess, even in populations living in endemic zones of amoebiasis carrying antibodies against amoebas. In conclusion, ELISA-IC:MC presented better diagnostic parameters than IHA although a negative serologic test does not rule out acute invasive amoebiasis.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Echinococcus/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/normas , Abscesso Hepático Amebiano/diagnóstico , Animais , Estudos de Casos e Controles , Echinococcus/imunologia , Inibidores Enzimáticos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Isotipos de Imunoglobulinas/sangue , Abscesso Hepático Amebiano/imunologia , Preservação Biológica , Fatores de TempoRESUMO
Angiogenesis is fundamental to tumorigenesis and an attractive target for therapeutic intervention against cancer. We have recently demonstrated that CD13 (aminopeptidase N) expressed by nonmalignant host cells of unspecified types regulate tumor blood vessel development. Here, we compare CD13 wild-type and null bone marrow-transplanted tumor-bearing mice to show that host CD13(+) bone marrow-derived cells promote cancer progression via their effect on angiogenesis. Furthermore, we have identified CD11b(+)CD13(+) myeloid cells as the immune subpopulation directly regulating tumor blood vessel development. Finally, we show that these cells are specifically localized within the tumor microenvironment and produce proangiogenic soluble factors. Thus, CD11b(+)CD13(+) myeloid cells constitute a population of bone marrow-derived cells that promote tumor progression and metastasis and are potential candidates for the development of targeted antiangiogenic drugs.
Assuntos
Indutores da Angiogênese/metabolismo , Células da Medula Óssea/metabolismo , Antígenos CD13 , Células Mieloides/metabolismo , Neoplasias Experimentais/metabolismo , Neovascularização Patológica/metabolismo , Animais , Células da Medula Óssea/patologia , Antígeno CD11b , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células Mieloides/patologia , Metástase Neoplásica , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Neovascularização Patológica/patologiaRESUMO
Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.
Assuntos
Antígenos CD13/metabolismo , Neoplasias Pulmonares/enzimologia , Animais , Antígenos CD13/genética , Linhagem Celular Tumoral , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Through the actuation of vibronic modes in cell-membrane-associated aminocyanines, using near-infrared light, a distinct type of molecular mechanical action can be exploited to rapidly kill cells by necrosis. Vibronic-driven action (VDA) is distinct from both photodynamic therapy and photothermal therapy as its mechanical effect on the cell membrane is not abrogated by inhibitors of reactive oxygen species and it does not induce thermal killing. Subpicosecond concerted whole-molecule vibrations of VDA-induced mechanical disruption can be achieved using very low concentrations (500 nM) of aminocyanines or low doses of light (12 J cm-2, 80 mW cm-2 for 2.5 min), resulting in complete eradication of human melanoma cells in vitro. Also, 50% tumour-free efficacy in mouse models for melanoma was achieved. The molecules that destroy cell membranes through VDA have been termed molecular jackhammers because they undergo concerted whole-molecule vibrations. Given that a cell is unlikely to develop resistance to such molecular mechanical forces, molecular jackhammers present an alternative modality for inducing cancer cell death.
Assuntos
Melanoma , Fotoquimioterapia , Camundongos , Animais , Humanos , Melanoma/tratamento farmacológico , Fotoquimioterapia/métodos , Morte Celular , Espécies Reativas de Oxigênio/metabolismoRESUMO
The family observed in this study included affected males and asymptomatic females. The patients shared specific digital abnormalities including postaxial polydactyly, cutaneous syndactyly, and brachydactyly. In addition, the patients exhibited mild-to-moderate intellectual disability and short stature coupled with microbrachycephaly, scoliosis, and cerebellar and renal hypoplasia. No chromosomal alterations or copy number variations were found in the index case. The genetic linkage analysis, which focused on the X chromosome, and the haplotype analysis detected a ~15.74 Mb candidate region located at Xp11.4-p11.21 with a LOD score of 4.8. Additionally, half of the mothers showed skewed X-inactivation, while the other mothers exhibited random inactivation patterns. The candidate region includes 28 protein-encoding genes that have not yet been implicated in human disorders. We speculate that the observed phenotype is compatible with a monogenic disorder in which the mutant gene plays a significant role during embryonic development. Based on the patients' clinical features, image studies, pedigree, chromosome location, and X-inactivation studies in the mothers, we propose that this family has a novel, specific syndrome with an X-linked recessive mode of inheritance.
Assuntos
Anormalidades Múltiplas/genética , Cromossomos Humanos X , Nanismo/genética , Dedos/anormalidades , Deficiência Intelectual/genética , Dedos do Pé/anormalidades , Anormalidades Múltiplas/diagnóstico , Adulto , Mapeamento Cromossômico , Diagnóstico Diferencial , Nanismo/diagnóstico , Feminino , Genes Recessivos , Genes Ligados ao Cromossomo X , Haplótipos , Humanos , Deficiência Intelectual/diagnóstico , Escore Lod , Masculino , México , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Linhagem , Fenótipo , Inativação do Cromossomo X , Adulto JovemRESUMO
BACKGROUND: TP53, the most mutated gene in solid cancers, has a profound impact on most hallmarks of cancer. Somatic TP53 mutations occur in high frequencies in head and neck cancers, including oral squamous cell carcinoma (OSCC). Our study aims to understand the role of TP53 gain-of-function mutation in modulating the tumor immune microenvironment (TIME) in OSCC. METHODS: Short hairpin RNA knockdown of mutant p53R172H in syngeneic oral tumors demonstrated changes in tumor growth between immunocompetent and immunodeficient mice. HTG EdgeSeq targeted messenger RNA sequencing was used to analyze cytokine and immune cell markers in tumors with inactivated mutant p53R172H. Flow cytometry and multiplex immunofluorescence (mIF) confirmed the role of mutant p53R172H in the TIME. The gene expression of patients with OSCC was analyzed by CIBERSORT and mIF was used to validate the immune landscape at the protein level. RESULTS: Mutant p53R172H contributes to a cytokine transcriptome network that inhibits the infiltration of cytotoxic CD8+ T cells and promotes intratumoral recruitment of regulatory T cells and M2 macrophages. Moreover, p53R172H also regulates the spatial distribution of immunocyte populations, and their distribution between central and peripheral intratumoral locations. Interestingly, p53R172H-mutated tumors are infiltrated with CD8+ and CD4+ T cells expressing programmed cell death protein 1, and these tumors responded to immune checkpoint inhibitor and stimulator of interferon gene 1 agonist therapy. CIBERSORT analysis of human OSCC samples revealed associations between immune cell populations and the TP53R175H mutation, which paralleled the findings from our syngeneic mouse tumor model. CONCLUSIONS: These findings demonstrate that syngeneic tumors bearing the TP53R172H gain-of-function mutation modulate the TIME to evade tumor immunity, leading to tumor progression and decreased survival.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Microambiente Tumoral , Proteína Supressora de Tumor p53 , Animais , Humanos , Camundongos , Carcinoma de Células Escamosas/genética , Linfócitos T CD8-Positivos , Citocinas , Modelos Animais de Doenças , Mutação com Ganho de Função , Neoplasias Bucais/genética , Mutação , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: The authors conducted a 2-year retrospective follow-up to investigate the efficiency of an extraforaminal full-endoscopic approach with foraminoplasty used to treat lateral compressive diseases of the lumbar spine in 247 patients. METHODS: The visual analogue scale (VAS), Oswestry disability index (ODI), and MacNab scale were used to analyze the results collected during the preoperative and postoperative periods. RESULTS: The most common diagnosis was disk herniation with lateral recess stenosis, and the most common surgical level among patients was between L4 and L5 on the left side. Pain decreased over time, as determined during sessions held to evaluate pain in the lumbar, gluteal, led, and foot regions. The ODI demonstrated significant enhancement over the evaluation period and the MacNab scale classified the surgery as good or excellent. The most common complication was dysesthesia. CONCLUSIONS: An extraforaminal full-endoscopic approach with foraminoplasty can be recommended in cases of lateral herniation or stenosis for patients with symptoms of radiculopathy, and for those who have not responded to conventional rehabilitation treatment or chronic pain management. Few complications arose as a result of this approach, and most of them were treated clinically.
RESUMO
Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of beta(1) integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein-protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Citoplasma/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteínas Nucleares/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Modelos Biológicos , Dados de Sequência Molecular , Transplante de Neoplasias , Proteínas Nucleares/química , Domínios de Homologia de srcRESUMO
Since its inception in 1975, the hybridoma technology revolutionized science and medicine, facilitating discoveries in almost any field from the laboratory to the clinic. Many technological advancements have been developed since then, to create these "magical bullets." Phage and yeast display libraries expressing the variable heavy and light domains of antibodies, single B-cell cloning from immunized animals of different species including humans or in silico approaches, all have rendered a myriad of newly developed antibodies or improved design of existing ones. However, still the majority of these antibodies or their recombinant versions are from hybridoma origin, a preferred methodology that trespass species barriers, due to the preservation of the natural functions of immune cells in producing the humoral response: antigen specific immunoglobulins. Remarkably, this methodology can be reproduced in small laboratories without the need of sophisticate equipment. In this chapter, we will describe the most recent methods utilized by our Monoclonal Antibodies Core Facility at the University of Texas-M.D. Anderson Cancer Center. During the last 10 years, the methods, techniques, and expertise implemented in our core had generated more than 350 antibodies for various applications.
Assuntos
Anticorpos Monoclonais , Linfócitos B , Animais , Anticorpos Monoclonais/genética , Antígenos , Hibridomas , TecnologiaRESUMO
Oral premalignant lesions (OPLs) are the precursors to oral cavity cancers, and have variable rates of progression to invasive disease. As an intermediate state, OPLs have acquired a subset of the genomic alterations while arising in an oral inflammatory environment. These specific genomic changes may facilitate the transition to an immune microenvironment that permits malignant transformation. Here, we will discuss mechanisms by which OPLs develop an immunosuppressive microenvironment that facilitates progression to invasive cancer. We will describe how genomic alterations and immune microenvironmental changes co-evolve and cooperate to promote OSCC progression. Finally, we will describe how these immune microenvironmental changes provide specific and unique evolutionary vulnerabilities for targeted therapies. Therefore, understanding the genomic changes that drive immunosuppressive microenvironments may eventually translate into novel biomarker and/or therapeutic approaches to limit the progression of OPLs to potential lethal oral cancers.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Bucais/genética , Lesões Pré-Cancerosas/genética , Microambiente Tumoral/genética , Carcinoma de Células Escamosas/patologia , Humanos , Neoplasias Bucais/patologia , Lesões Pré-Cancerosas/patologiaRESUMO
BACKGROUND: Many studies have emphasized the importance of interface contact between implants and the vertebral endplate (VE). The goal of this study was to analyze the shape and other specific parameters of the VE to provide reference data for better implant interface contact in intervertebral disc space procedures. METHODS: Cervical, thoracic, and lumbar spine midsagittal plane magnetic resonance images of 100 adults (58 women) were analyzed. The morphology of the VEs was classified as concave, convex, flat, or irregular. Midsagittal endplate length (ML), endplate concavity depth (ECD), and endplate concavity axis (ECA) location were measured in the midsagittal plane. The parameters were compared between the cervical, thoracic, and lumbar spines and between the sexes. RESULTS: The VE morphology, ML, ECD, and ECA showed variations along the spine, mainly in the cervical and lower lumbar spines. The sagittal geometry of the VE was not flat or uniform along the cervical, thoracic, and lumbar spines. Different morphological types were observed along different spinal segments and according to sex. In the cervical spine, the majority of cranial VEs were flat, while caudal VEs were mostly concave. CONCLUSION: Sagittal VE geometry should be taken into consideration during the use of intervertebral cages or disc arthroplasty.