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1.
Gastroenterology ; 163(5): 1228-1241, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35870513

RESUMO

BACKGROUND & AIMS: Mechanisms contributing to the onset and progression of Barrett's (BE)-associated esophageal adenocarcinoma (EAC) remain elusive. Here, we interrogated the major signaling pathways deregulated early in the development of Barrett's neoplasia. METHODS: Whole-transcriptome RNA sequencing analysis was performed in primary BE, EAC, normal esophageal squamous, and gastric biopsy tissues (n = 89). Select pathway components were confirmed by quantitative polymerase chain reaction in an independent cohort of premalignant and malignant biopsy tissues (n = 885). Functional impact of selected pathway was interrogated using transcriptomic, proteomic, and pharmacogenetic analyses in mammalian esophageal organotypic and patient-derived BE/EAC cell line models, in vitro and/or in vivo. RESULTS: The vast majority of primary BE/EAC tissues and cell line models showed hyperactivation of EphB2 signaling. Transcriptomic/proteomic analyses identified EphB2 as an endogenous binding partner of MYC binding protein 2, and an upstream regulator of c-MYC. Knockdown of EphB2 significantly impeded the viability/proliferation of EAC and BE cells in vitro/in vivo. Activation of EphB2 in normal esophageal squamous 3-dimensional organotypes disrupted epithelial maturation and promoted columnar differentiation programs, notably including MYC. EphB2 and MYC showed selective induction in esophageal submucosal glands with acinar ductal metaplasia, and in a porcine model of BE-like esophageal submucosal gland spheroids. Clinically approved inhibitors of MEK, a protein kinase that regulates MYC, effectively suppressed EAC tumor growth in vivo. CONCLUSIONS: The EphB2 signaling is frequently hyperactivated across the BE-EAC continuum. EphB2 is an upstream regulator of MYC, and activation of EphB2-MYC axis likely precedes BE development. Targeting EphB2/MYC could be a promising therapeutic strategy for this often refractory and aggressive cancer.


Assuntos
Esôfago de Barrett , Carcinoma de Células Escamosas , Neoplasias Esofágicas , Suínos , Animais , Esôfago de Barrett/patologia , Efrina-B2/genética , Proteômica , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas/patologia , Proto-Oncogenes , Proteínas Tirosina Quinases/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Mamíferos/genética
2.
Gastroenterology ; 156(6): 1761-1774, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30768984

RESUMO

BACKGROUND & AIMS: Esophageal adenocarcinoma (EAC) is resistant to standard chemoradiation treatments, and few targeted therapies are available. We used large-scale tissue profiling and pharmacogenetic analyses to identify deregulated signaling pathways in EAC tissues that might be targeted to slow tumor growth or progression. METHODS: We collected 397 biopsy specimens from patients with EAC and nonmalignant Barrett's esophagus (BE), with or without dysplasia. We performed RNA-sequencing analyses and used systems biology approaches to identify pathways that are differentially activated in EAC vs nonmalignant dysplastic tissues; pathway activities were confirmed with immunohistochemistry and quantitative real-time polymerase chain reaction analyses of signaling components in patient tissue samples. Human EAC (FLO-1 and EsoAd1), dysplastic BE (CP-B, CP-C, CP-D), and nondysplastic BE (CP-A) cells were incubated with pharmacologic inhibitors or transfected with small interfering RNAs. We measured effects on proliferation, colony formation, migration, and/or growth of xenograft tumors in nude mice. RESULTS: Comparisons of EAC vs nondysplastic BE tissues showed hyperactivation of transforming growth factor-ß (TGFB) and/or Jun N-terminal kinase (JNK) signaling pathways in more than 80% of EAC samples. Immunohistochemical analyses showed increased nuclear localization of phosphorylated JUN and SMAD proteins in EAC tumor tissues compared with nonmalignant tissues. Genes regulated by the TGFB and JNK pathway were overexpressed specifically in EAC and dysplastic BE. Pharmacologic inhibition or knockdown of TGFB or JNK signaling components in EAC cells (FLO-1 or EsoAd1) significantly reduced cell proliferation, colony formation, cell migration, and/or growth of xenograft tumors in mice in a SMAD4-independent manner. Inhibition of the TGFB pathway in BE cell lines reduced the proliferation of dysplastic, but not nondysplastic, cells. CONCLUSIONS: In a transcriptome analysis of EAC and nondysplastic BE tissues, we found the TGFB and JNK signaling pathways to be hyperactivated in EACs and the genes regulated by these pathways to be overexpressed in EAC and dysplastic BE. Inhibiting these pathways in EAC cells reduces their proliferation, migration, and formation of xenograft tumors. Strategies to block the TGFB and JNK signaling pathways might be developed for treatment of EAC.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Sistema de Sinalização das MAP Quinases/genética , RNA Neoplásico/análise , Fator de Crescimento Transformador beta/metabolismo , Animais , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Benzamidas/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Dioxóis/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Testes Farmacogenômicos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Pirazóis/farmacologia , Quinolinas/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Proteínas Smad/genética , Proteínas Smad/metabolismo , Biologia de Sistemas , Transcriptoma , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética , Ensaio Tumoral de Célula-Tronco
3.
Hum Mutat ; 39(8): 1092-1101, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29749045

RESUMO

Characterizing moderate penetrance susceptibility genes is an emerging frontier in colorectal cancer (CRC) research. GALNT12 is a strong candidate CRC-susceptibility gene given previous linkage and association studies, and inactivating somatic and germline alleles in CRC patients. Previously, we found rare segregating germline GALNT12 variants in a clinic-based cohort (N = 118) with predisposition for CRC. Here, we screened a new population-based cohort of incident CRC cases (N = 479) for rare (MAF ≤1%) deleterious germline GALNT12 variants. GALNT12 screening revealed eight rare variants. Two variants were previously described (p.Asp303Asn, p.Arg297Trp), and additionally, we found six other rare variants: five missense (p.His101Gln, p.Ile142Thr, p.Glu239Gln, p.Thr286Met, p.Val290Phe) and one putative splice-altering variant (c.732-8 G>T). Sequencing of population-matched controls (N = 400) revealed higher burden of these variants in CRC cases compared with healthy controls (P = 0.0381). We then functionally characterized the impact of substitutions on GALNT12 enzyme activity using in vitro-derived peptide substrates. Three of the newly identified GALNT12 missense variants (p.His101Gln, p.Ile142Thr, p.Val290Phe) demonstrated a marked loss (>2-fold reduction) of enzymatic activity compared with wild-type (P ≤ 0.05), whereas p.Glu239Gln exhibited a ∼2-fold reduction in activity (P = 0.077). These findings provide strong, independent evidence for the association of GALNT12 defects with CRC-susceptibility; underscoring implications for glycosylation pathway defects in CRC.


Assuntos
Neoplasias Colorretais/genética , N-Acetilgalactosaminiltransferases/genética , Western Blotting , Linhagem Celular Tumoral , Predisposição Genética para Doença/genética , Genótipo , Humanos , Repetições de Microssatélites/genética , Proteínas Recombinantes/genética
5.
Cancer Res ; 76(19): 5628-5633, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27503924

RESUMO

Esophageal adenocarcinoma is a deadly cancer with increasing incidence in the United States, but mechanisms underlying pathogenesis are still mostly elusive. In addressing this question, we assessed gene fusion landscapes by comprehensive RNA sequencing (RNAseq) of 55 pretreatment esophageal adenocarcinoma and 49 nonmalignant biopsy tissues from patients undergoing endoscopy for Barrett's esophagus. In this cohort, we identified 21 novel candidate esophageal adenocarcinoma-associated fusions occurring in 3.33% to 11.67% of esophageal adenocarcinomas. Two candidate fusions were selected for validation by PCR and Sanger sequencing in an independent set of pretreatment esophageal adenocarcinoma (N = 115) and nonmalignant (N = 183) biopsy tissues. In particular, we observed RPS6KB1-VMP1 gene fusion as a recurrent event occurring in approximately 10% of esophageal adenocarcinoma cases. Notably, esophageal adenocarcinoma cases harboring RPS6KB1-VMP1 fusions exhibited significantly poorer overall survival as compared with fusion-negative cases. Mechanistic investigations established that the RPS6KB1-VMP1 transcript coded for a fusion protein, which significantly enhanced the growth rate of nondysplastic Barrett's esophagus cells. Compared with the wild-type VMP1 protein, which mediates normal cellular autophagy, RPS6KB1-VMP1 fusion exhibited aberrant subcellular localization and was relatively ineffective in triggering autophagy. Overall, our findings identified RPS6KB1-VMP1 as a genetic fusion that promotes esophageal adenocarcinoma by modulating autophagy-related processes, offering new insights into the molecular pathogenesis of esophageal adenocarcinomas. Cancer Res; 76(19); 5628-33. ©2016 AACR.


Assuntos
Adenocarcinoma/genética , Neoplasias Esofágicas/genética , Fusão Gênica , Proteínas de Membrana/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Análise de Sequência de RNA , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Autofagia , Linhagem Celular Tumoral , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Humanos
6.
Sci Rep ; 6: 23642, 2016 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-27004849

RESUMO

The molecular basis of aberrant protein glycosylation, a pathological alteration widespread in colorectal cancers (CRC), and the mechanisms by which it contributes to tumor progression remain largely unknown. We performed targeted re-sequencing of 430 glycosylation-associated genes in a series of patient-derived CRC cell lines (N = 31) and matched primary tumor tissues, identifying 12 new significantly mutated glycosylation-associated genes in colon cancer. In particular, we observed an enrichment of mutations in genes (B3GNT2, B4GALT2, ST6GALNAC2) involved in the biosynthesis of N- and Cores 1-3 O-linked glycans in the colon, accounting for ~16% of the CRCs tested. Analysis of independent large-scale tumor tissue datasets confirmed recurrent mutations within these genes in colon and other gastrointestinal cancers. Systematic biochemical and phenotypic characterization of the candidate wild-type and mutant glycosyltransferases demonstrated these mutations as either markedly altering protein localization, post-translational modification, encoded enzymatic activities and/or the migratory potential of colon carcinoma cells. These findings suggest that functionally deleterious mutations in glycosyltransferase genes in part underlie aberrant glycosylation, and contribute to the pathogenesis of molecular subsets of colon and other gastrointestinal malignancies.


Assuntos
Neoplasias do Colo/genética , Análise Mutacional de DNA/métodos , Galactosiltransferases/genética , Glicosiltransferases/genética , N-Acetilglucosaminiltransferases/genética , Sialiltransferases/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Glicosilação , Humanos , Polissacarídeos/biossíntese , Processamento de Proteína Pós-Traducional
7.
Biochem Biophys Res Commun ; 417(3): 1058-62, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22222369

RESUMO

LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Núcleo Celular/enzimologia , Regulação Leucêmica da Expressão Gênica , Proteínas com Domínio LIM/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Ativação Transcricional , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética
8.
Cell Signal ; 23(7): 1170-8, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21397011

RESUMO

STAT (signal transducer and activator of transcription) proteins play a critical role in cellular response to a wide variety of cytokines and growth factors by regulating specific nuclear genes. STAT-dependent gene transcription can be finely tuned through the association with co-factors in the nucleus. We showed previously that STAT5 (including 5a and 5b) specifically interacts with a mitochondrial enzyme PDC-E2 (E2 subunit of pyruvate dehydrogenase complex) in both leukemic T cells and cytokine-stimulated cells. However, the functional significance of this novel association remains largely unknown. Here we report that PDC-E2 may function as a co-activator in STAT5-dependent nuclear gene expression. Subcellular fractionation analysis revealed that a substantial amount of PDC-E2 was constitutively present in the nucleus of BaF3, an interleukin-3 (IL-3)-dependent cell line. IL-3-induced tyrosine-phosphorylated STAT5 associated with nuclear PDC-E2 in co-immunoprecipitation analysis. These findings were confirmed by confocal immunofluorescence microscopy showing constant nuclear localization of PDC-E2 and its co-localization with STAT5 after IL-3 stimulation. Similar to mitochondrial PDC-E2, nuclear PDC-E2 was lipoylated and associated with PDC-E1. Overexpression of PDC-E2 in BaF3 cells augmented IL-3-induced STAT5 activity as measured by reporter assay with consensus STAT5-binding sites. Consistent with the reporter data, PDC-E2 overexpression in BaF3 cells led to elevated mRNA levels of endogenous SOCS3 (suppressor of cytokine signaling 3) gene, a known STAT5 target. We further identified two functional STAT5-binding sites in the SOCS3 gene promoter important for its IL-3-inducibility. The observation that both cis-acting elements were essential to detect the stimulatory effect by PDC-E2 strongly supports the role of PDC-E2 in up-regulating the transactivating ability of STAT5. All together, our results reveal a novel function of PDC-E2 in the nucleus. It also raises the possibility of nuclear-mitochondrial crosstalk through the interaction between STAT5 and PDC-E2.


Assuntos
Núcleo Celular/enzimologia , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/metabolismo , Mitocôndrias/enzimologia , Fator de Transcrição STAT5/metabolismo , Animais , Linhagem Celular , Genes Reporter , Imunoprecipitação , Interleucina-3/farmacologia , Lipoilação , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Transcrição Gênica
9.
Oncol Rep ; 25(3): 677-83, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21234523

RESUMO

Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here, we report that among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identified the positive regulatory phosphotyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases.


Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Mutação/fisiologia , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fosfotransferases/química , Fosfotransferases/genética , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Mapeamento de Interação de Proteínas , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/genética , Transfecção , Tirosina/genética
10.
Int J Oncol ; 36(5): 1201-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20372794

RESUMO

Lck is an Src family protein tyrosine kinase with predominant T cell expression. Aberrant expression or activation of Lck kinase has been reported in both lymphoid and non-lymphoid malignancies. We showed previously that the signal transduction pathway involving Janus kinase (JAK) and signal transducer and activator of transcription (STAT) is constitutively activated and contributes to Lck-mediated oncogenesis. Under normal physiological conditions, active STAT proteins induce the expression of suppressor of cytokine signaling (SOCS) family proteins to inhibit further JAK/STAT signaling. It is not fully understood whether and how SOCS-mediated negative feedback control is dysregulated in Lck-transformed cells. Here we report that two SOCS family members, SOCS1 and SOCS3, are not expressed in Lck-transformed LSTRA leukemia. While SOCS1 gene is silenced by DNA hypermethylation, loss of SOCS3 expression is through a mechanism independent of epigenetic silencing by DNA methylation. Furthermore, ectopic expression of SOCS1 or SOCS3 leads to reduced cell proliferation and increased apoptosis in Lck-transformed cells. This is consistent with the attenuation of Lck kinase activity by exogenous SOCS1 or SOCS3 expression. Downstream STAT5 activity is also inhibited as shown by reduced STAT5 tyrosine phosphorylation and in vitro DNA binding. All together, our data highlight the importance of silencing multiple SOCS genes in tumorigenesis and support the roles of SOCS1 and SOCS3 as tumor suppressors toward oncogenic Lck kinase.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Transformação Celular Neoplásica , Metilação de DNA , Epigênese Genética , Humanos , Camundongos , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas
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