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1.
Gut ; 72(4): 624-637, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36002248

RESUMEN

OBJECTIVE: Many cancers engage embryonic genes for rapid growth and evading the immune system. SOX9 has been upregulated in many tumours, yet the role of SOX9 in mediating immunosuppressive tumour microenvironment is unclear. Here, we aim to dissect the role of SOX9-mediated cancer stemness attributes and immunosuppressive microenvironment in advanced gastric adenocarcinoma (GAC) for novel therapeutic discoveries. METHODS: Bulk RNAseq/scRNA-seq, patient-derived cells/models and extensive functional studies were used to identify the expression and functions of SOX9 and its target genes in vitro and in vivo. Immune responses were studied in PBMCs or CD45+ immune cells cocultured with tumour cells with SOX9high or knockout and the KP-Luc2 syngeneic models were used for efficacy of combinations. RESULTS: SOX9 is one of the most upregulated SOX genes in GAC and highly expressed in primary and metastatic tissues and associated with poor prognosis. Depletion of SOX9 in patient-derived GAC cells significantly decreased cancer stemness attributes, tumour formation and metastases and consistently increased CD8+ T cell responses when cocultured with PBMCs/CD45+ cells from GAC patients. RNA sequencing identified the leukaemia inhibitory factor (LIF) as the top secreted molecule regulated by SOX9 in tumour cells and was enriched in malignant ascites and mediated SOX9-induced M2 macrophage repolarisation and inhibited T cell function. CONCLUSION: Epithelial SOX9 is critical in suppressing CD8+ T cell responses and modified macrophage function in GAC through the paracrine LIF factor. Cotargeting LIF/LIFR and CSF1R has great potential in targeting SOX9-mediated cancer stemness, T cell immunosuppression and metastases suggesting the novel combination therapy against advanced GAC.


Asunto(s)
Adenocarcinoma , Neoplasias Gástricas , Humanos , Microambiente Tumoral , Adenocarcinoma/genética , Adenocarcinoma/patología , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Inmunosupresores , Terapia de Inmunosupresión , Factor de Transcripción SOX9/genética
2.
Gut ; 71(3): 467-478, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-33785559

RESUMEN

BACKGROUND: Gastric adenocarcinoma (GAC) is a lethal disease with limited therapeutic options. Genetic alterations in chromatin remodelling gene AT-rich interactive domain 1A (ARID1A) and mTOR pathway activation occur frequently in GAC. Targeting the mechanistic target of rapamycin (mTOR) pathway in unselected patients has failed to show survival benefit. A deeper understanding of GAC might identify a subset that can benefit from mTOR inhibition. METHODS: Genomic alterations in ARID1A were analysed in GAC. Mouse gastric epithelial cells from CK19-Cre-Arid1Afl/fl and wild-type mice were used to determine the activation of oncogenic genes due to loss of Arid1A. Functional studies were performed to determine the significance of loss of ARID1A and the sensitivity of ARID1A-deficient cancer cells to mTOR inhibition in GAC. RESULTS: More than 30% of GAC cases had alterations (mutations or deletions) of ARID1A and ARID1A expression was negatively associated with phosphorylation of S6 and SOX9 in GAC tissues and patient-derived xenografts (PDXs). Activation of mTOR signalling (increased pS6) and SOX9 nuclear expression were strongly increased in Arid1A-/- mouse gastric tissues which could be curtailed by RAD001, an mTOR inhibitor. Knockdown of ARID1A in GAC cell lines increased pS6 and nuclear SOX9 and increased sensitivity to an mTOR inhibitor which was further amplified by its combination with fluorouracil both in vitro and in vivo in PDXs. CONCLUSIONS: The loss of ARID1A activates pS6 and SOX9 in GAC, which can be effectively targeted by an mTOR inhibitor. Therefore, our studies suggest a new therapeutic strategy of clinically targeting the mTOR pathway in patients with GAC with ARID1A deficiency.


Asunto(s)
Adenocarcinoma/etiología , Proteínas de Unión al ADN/fisiología , Factor de Transcripción SOX9/fisiología , Transducción de Señal/fisiología , Neoplasias Gástricas/etiología , Serina-Treonina Quinasas TOR/fisiología , Factores de Transcripción/fisiología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular , Ratones , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología
3.
Gut ; 70(12): 2238-2248, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-33487592

RESUMEN

OBJECTIVE: Gastro-oesophageal cancers (GEC) are resistant to therapy and lead to poor prognosis. The cancer stem cells (CSCs) and antiapoptotic pathways often confer therapy resistance. We sought to elucidate the antitumour action of a BCL-2 inhibitor, AT101 in GEC in vitro, in vivo and in a clinical trial. METHODS: Extensive preclinical studies in vitro and in vivo were carried out to establish the mechanism action of AT101 on targeting CSCs and antiapoptotic proteins. A pilot clinical trial in patients with GEC was completed with AT-101 added to standard chemoradiation. RESULTS: Overexpression of BCL-2 and MCL-1 was noted in gastric cancer tissues (GC). AT-101 induced apoptosis, reduced proliferation and tumour sphere formation in MCL-1/BCL-2 high GC cells. Interestingly, AT101 dramatically downregulated genes (YAP-1/Sox9) that control CSCs in GEC cell lines regardless of BCL-2/MCL-1 expression. Addition of docetaxel to AT-101 amplified its antiproliferation and induced apoptosis effects. In vivo studies confirmed the combination of AT101 and docetaxel demonstrated stronger antitumour activity accompanied with significant decrease of CSCs biomarkers (YAP1/SOX9). In a pilot clinical trial, 13 patients with oesophageal cancer (EC) received AT101 orally concurrently with chemoradiation. We observed dramatic clinical complete responses and encouraging overall survival in these patients. Clinical specimen analyses revealed that AT-101 dramatically reduced the expression of CSCs genes in treated EC specimens indicating antitumour activity of AT101 relies more on its anti-CSCs activity. CONCLUSIONS: Our preclinical and clinical data suggest that AT-101 overcomes resistance by targeting CSCs pathways suggesting a novel mechanism of action of AT101 in patients with GEC.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Neoplasias Esofágicas/tratamiento farmacológico , Gosipol/análogos & derivados , Células Madre Neoplásicas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Neoplasias Gástricas/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Docetaxel/farmacología , Neoplasias Esofágicas/genética , Femenino , Gosipol/farmacología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proyectos Piloto , Proteínas Proto-Oncogénicas c-bcl-2/genética , Neoplasias Gástricas/genética
4.
Gut ; 70(1): 55-66, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32345613

RESUMEN

OBJECTIVE: Peritoneal carcinomatosis (PC; malignant ascites or implants) occurs in approximately 45% of advanced gastric adenocarcinoma (GAC) patients and associated with a poor survival. The molecular events leading to PC are unknown. The yes-associated protein 1 (YAP1) oncogene has emerged in many tumour types, but its clinical significance in PC is unclear. Here, we investigated the role of YAP1 in PC and its potential as a therapeutic target. METHODS: Patient-derived PC cells, patient-derived xenograft (PDX) and patient-derived orthotopic (PDO) models were used to study the function of YAP1 in vitro and in vivo. Immunofluorescence and immunohistochemical staining, RNA sequencing (RNA-Seq) and single-cell RNA-Seq (sc-RNA-Seq) were used to elucidate the expression of YAP1 and PC cell heterogeneity. LentiCRISPR/Cas9 knockout of YAP1 and a YAP1 inhibitor were used to dissect its role in PC metastases. RESULTS: YAP1 was highly upregulated in PC tumour cells, conferred cancer stem cell (CSC) properties and appeared to be a metastatic driver. Dual staining of YAP1/EpCAM and sc-RNA-Seq revealed that PC tumour cells were highly heterogeneous, YAP1high PC cells had CSC-like properties and easily formed PDX/PDO tumours but also formed PC in mice, while genetic knockout YAP1 significantly slowed tumour growth and eliminated PC in PDO model. Additionally, pharmacologic inhibition of YAP1 specifically reduced CSC-like properties and suppressed tumour growth in YAP1high PC cells especially in combination with cytotoxics in vivo PDX model. CONCLUSIONS: YAP1 is essential for PC that is attenuated by YAP1 inhibition. Our data provide a strong rationale to target YAP1 in clinic for GAC patients with PC.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Adenocarcinoma/secundario , Neoplasias Peritoneales/secundario , Neoplasias Gástricas/patología , Animales , Técnicas de Cultivo de Célula , Humanos , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Señalizadoras YAP
5.
Gut ; 69(1): 18-31, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31171626

RESUMEN

OBJECTIVE: Peritoneal carcinomatosis (PC) occurs frequently in patients with gastric adenocarcinoma (GAC) and confers a poor prognosis. Multiplex profiling of primary GACs has been insightful but the underpinnings of PC's development/progression remain largely unknown. We characterised exome/transcriptome/immune landscapes of PC cells from patients with GAC aiming to identify novel therapeutic targets. DESIGN: We performed whole-exome sequencing (WES) and whole transcriptome sequencing (RNA-seq) on 44 PC specimens (43 patients with PC) including an integrative analysis of WES, RNA-seq, immune profile, clinical and pathological phenotypes to dissect the molecular pathogenesis, identifying actionable targets and/or biomarkers and comparison with TCGA primary GACs. RESULTS: We identified distinct alterations in PC versus primary GACs, such as more frequent CDH1 and TAF1 mutations, 6q loss and chr19 gain. Alterations associated with aggressive PC phenotypes emerged with increased mutations in TP53, CDH1, TAF1 and KMT2C, higher level of 'clock-like' mutational signature, increase in whole-genome doublings, chromosomal instability (particularly, copy number losses), reprogrammed microenvironment, enriched cell cycle pathways, MYC activation and impaired immune response. Integrated analysis identified two main molecular subtypes: 'mesenchymal-like' and 'epithelial-like' with discriminating response to chemotherapy (31% vs 71%). Patients with the less responsive 'mesenchymal-like' subtype had high expression of immune checkpoint T-Cell Immunoglobulin And Mucin Domain-Containing Protein 3 (TIM-3), its ligand galectin-9, V-domain Ig suppressor of T cell activation (VISTA) and transforming growth factor-ß as potential therapeutic immune targets. CONCLUSIONS: We have uncovered the unique mutational landscape, copy number alteration and gene expression profile of PC cells and defined PC molecular subtypes, which correlated with PC therapy resistance/response. Novel targets and immune checkpoint proteins have been identified with a potential to be translated into clinics.


Asunto(s)
Adenocarcinoma/secundario , Neoplasias Peritoneales/secundario , Neoplasias Gástricas/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Inestabilidad Cromosómica , Variaciones en el Número de Copia de ADN/genética , ADN de Neoplasias/genética , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida/métodos , Mutación , Neoplasias Peritoneales/tratamiento farmacológico , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/inmunología , Ploidias , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/inmunología , Secuenciación del Exoma/métodos
6.
Mol Cancer ; 18(1): 141, 2019 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-31601234

RESUMEN

BACKGROUND: PVT1 has emerged as an oncogene in many tumor types. However, its role in Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) is unknown. The aim of this study was to assess the role of PVT1 in BE/EAC progression and uncover its therapeutic value against EAC. METHODS: PVT1 expression was assessed by qPCR in normal, BE, and EAC tissues and statistical analysis was performed to determine the association of PVT1 expression and EAC (stage, metastases, and survival). PVT1 antisense oligonucleotides (ASOs) were tested for their antitumor activity in vitro and in vivo. RESULTS: PVT1 expression was up-regulated in EACs compared with paired BEs, and normal esophageal tissues. High expression of PVT1 was associated with poor differentiation, lymph node metastases, and shorter survival. Effective knockdown of PVT1 in EAC cells using PVT1 ASOs resulted in decreased cell proliferation, invasion, colony formation, tumor sphere formation, and reduced proportion of ALDH1A1+ cells. Mechanistically, we discovered mutual regulation of PVT1 and YAP1 in EAC cells. Inhibition of PVT1 by PVT1 ASOs suppressed YAP1 expression through increased phosphor-LATS1and phosphor-YAP1 while knockout of YAP1 in EAC cells significantly suppressed PVT1 levels indicating a positive regulation of PVT1 by YAP1. Most importantly, we found that targeting both PVT1 and YAP1 using their specific ASOs led to better antitumor activity in vitro and in vivo. CONCLUSIONS: Our results provide strong evidence that PVT1 confers an aggressive phenotype to EAC and is a poor prognosticator. Combined targeting of PVT1 and YAP1 provided the highest therapeutic index and represents a novel therapeutic strategy.


Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/mortalidad , Biomarcadores de Tumor , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidad , Regulación Neoplásica de la Expresión Génica , ARN Largo no Codificante/genética , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Adenocarcinoma/tratamiento farmacológico , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Modelos Animales de Enfermedad , Neoplasias Esofágicas/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Modelos Biológicos , Pronóstico , Factores de Transcripción/antagonistas & inhibidores , Proteínas Señalizadoras YAP
7.
Br J Cancer ; 118(1): 52-61, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29136404

RESUMEN

BACKGROUND: Overexpression of Galectin-3 (Gal-3), a ß-galactoside binding protein, has been noted in many tumour types but its functional significance and clinical utility in gastric adenocarcinoma (GAC) are not well known. METHODS: We studied 184 GAC patients characterised by histologic grade, sub-phenotypes (diffuse vs intestinal), and ethnicity (Asians vs North Americans). Immunohistochemistry was performed to assess the expression of Gal-3 in human GACs and we correlated it to the clinical outcomes. Cell proliferation, invasion, co-immunoprecipitation and kinase activity assays were done in genetically stable Gal-3 overexpressing GC cell lines and the parental counterparts to delineate the mechanisms of action and activity of inhibitors. RESULTS: Most patients were men, Asian, and had a poorly differentiated GAC. Gal-3 was over-expressed in poorly differentiated (P=0.002) tumours and also in diffuse sub-phenotype (P=0.02). Gal-3 overexpression was associated with shorter overall survival (OS; P=0.026) in all patients. Although, Gal-3 over-expression was not prognostic in the Asian cohort (P=0.337), it was highly prognostic in the North American cohort (P=0.001). In a multivariate analysis, Gal-3 (P=0.001) and N-stage (P=<0.001) were independently prognostic for shorter OS. Mechanistically, Gal-3 induced c-MYC expression through increasing RalA activity and an enhanced YAP1/RalA/RalBP complex to confer an aggressive phenotype. YAP1/BET bromodomain inhibitors reduced Gal-3-mediated aggressive phenotypes in GAC cells. CONCLUSIONS: Gal-3 is an independent prognostic marker of shorter OS and a novel therapeutic target particularly in diffuse type GAC in North American patients.


Asunto(s)
Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Neoplasias Gástricas/patología , Regulación hacia Arriba , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Azepinas/farmacología , Proteínas Sanguíneas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Galectinas , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Clasificación del Tumor , Fenotipo , Fosfoproteínas/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-myc/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Factores de Transcripción , Triazoles/farmacología , Proteínas Señalizadoras YAP , Proteínas de Unión al GTP ral/metabolismo
8.
Br J Cancer ; 117(5): 648-655, 2017 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-28728163

RESUMEN

BACKGROUND: Predictive biomarkers or signature(s) for oesophageal cancer (OC) patients undergoing preoperative therapy could help administration of effective therapy, avoidance of ineffective ones, and establishment new strategies. Since the hedgehog pathway is often upregulated in OC, we examined its transcriptional factor, Gli-1, which confers therapy resistance, we wanted to assess Gli-1 as a predictive biomarker for chemoradiation response and validate it. METHODS: Untreated OC tissues from patients who underwent chemoradiation and surgery were assessed for nuclear Gli-1 by immunohistochemistry and labelling indices (LIs) were correlated with pathologic complete response (pathCR) or

Asunto(s)
Adenocarcinoma/química , Adenocarcinoma/terapia , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/terapia , Núcleo Celular/química , Quimioradioterapia Adyuvante , Neoplasias Esofágicas/química , Neoplasias Esofágicas/terapia , Proteína con Dedos de Zinc GLI1/análisis , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/análisis , Sistemas CRISPR-Cas , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos , Métodos Epidemiológicos , Neoplasias Esofágicas/patología , Esofagectomía , Femenino , Edición Génica , Proteínas Hedgehog/análisis , Proteínas Hedgehog/genética , Humanos , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante , ARN Mensajero/metabolismo , Tolerancia a Radiación , Proteína con Dedos de Zinc GLI1/antagonistas & inhibidores , Proteína con Dedos de Zinc GLI1/genética
10.
Cancer Res ; 83(22): 3726-3738, 2023 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-37738407

RESUMEN

The peritoneal cavity is a common site of gastric adenocarcinoma (GAC) metastasis. Peritoneal carcinomatosis (PC) is resistant to current therapies and confers poor prognosis, highlighting the need to identify new therapeutic targets. CD47 conveys a "don't eat me" signal to myeloid cells upon binding its receptor signal regulatory protein alpha (SIRPα), which helps tumor cells circumvent macrophage phagocytosis and evade innate immune responses. Previous studies demonstrated that the blockade of CD47 alone results in limited clinical benefits, suggesting that other target(s) might need to be inhibited simultaneously with CD47 to elicit a strong antitumor response. Here, we found that CD47 was highly expressed on malignant PC cells, and elevated CD47 was associated with poor prognosis. Galectin-3 (Gal3) expression correlated with CD47 expression, and coexpression of Gal3 and CD47 was significantly associated with diffuse type, poor differentiation, and tumor relapse. Depletion of Gal3 reduced expression of CD47 through inhibition of c-Myc binding to the CD47 promoter. Furthermore, injection of Gal3-deficient tumor cells into either wild-type and Lgals3-/- mice led to a reduction in M2 macrophages and increased T-cell responses compared with Gal3 wild-type tumor cells, indicating that tumor cell-derived Gal3 plays a more important role in GAC progression and phagocytosis than host-derived Gal3. Dual blockade of Gal3 and CD47 collaboratively suppressed tumor growth, increased phagocytosis, repolarized macrophages, and boosted T-cell immune responses. These data uncovered that Gal3 functions together with CD47 to suppress phagocytosis and orchestrate immunosuppression in GAC with PC, which supports exploring a novel combination therapy targeting Gal3 and CD47. SIGNIFICANCE: Dual inhibition of CD47 and Gal3 enhances tumor cell phagocytosis and reprograms macrophages to overcome the immunosuppressive microenvironment and suppress tumor growth in peritoneal metastasis of gastric adenocarcinoma.


Asunto(s)
Adenocarcinoma , Neoplasias , Neoplasias Peritoneales , Neoplasias Gástricas , Animales , Ratones , Antígenos de Diferenciación/metabolismo , Antígeno CD47/genética , Galectina 3/genética , Neoplasias/tratamiento farmacológico , Fagocitosis , Linfocitos T/metabolismo , Microambiente Tumoral
11.
Front Oncol ; 13: 1062424, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36865791

RESUMEN

Purpose: To establish a fast and accurate detection method for interspecies contaminations in the patient-derived xenograft (PDX) models and cell lines, and to elucidate possible mechanisms if interspecies oncogenic transformation is detected. Methods: A fast and highly sensitive intronic qPCR method detecting Gapdh intronic genomic copies was developed to quantify if cells were human or murine or a mixture. By this method, we documented that murine stromal cells were abundant in the PDXs; we also authenticated our cell lines to be human or murine. Results: In one mouse model, GA0825-PDX transformed murine stromal cells into a malignant tumorigenic murine P0825 cell line. We traced the timeline of this transformation and discovered three subpopulations descended from the same GA0825-PDX model: epithelium-like human H0825, fibroblast-like murine M0825, and main passaged murine P0825 displayed differences in tumorigenic capability in vivo. P0825 was the most aggressive and H0825 was weakly tumorigenic. Immunofluorescence (IF) staining revealed that P0825 cells highly expressed several oncogenic and cancer stem cell markers. Whole exosome sequencing (WES) analysis revealed that TP53 mutation in the human ascites IP116-generated GA0825-PDX may have played a role in the human-to-murine oncogenic transformation. Conclusion: This intronic qPCR is able to quantify human/mouse genomic copies with high sensitivity and within a time frame of a few hours. We are the first to use intronic genomic qPCR for authentication and quantification of biosamples. Human ascites transformed murine stroma into malignancy in a PDX model.

12.
Cancer Cell ; 41(8): 1407-1426.e9, 2023 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-37419119

RESUMEN

Understanding tumor microenvironment (TME) reprogramming in gastric adenocarcinoma (GAC) progression may uncover novel therapeutic targets. Here, we performed single-cell profiling of precancerous lesions, localized and metastatic GACs, identifying alterations in TME cell states and compositions as GAC progresses. Abundant IgA+ plasma cells exist in the premalignant microenvironment, whereas immunosuppressive myeloid and stromal subsets dominate late-stage GACs. We identified six TME ecotypes (EC1-6). EC1 is exclusive to blood, while EC4, EC5, and EC2 are highly enriched in uninvolved tissues, premalignant lesions, and metastases, respectively. EC3 and EC6, two distinct ecotypes in primary GACs, associate with histopathological and genomic characteristics, and survival outcomes. Extensive stromal remodeling occurs in GAC progression. High SDC2 expression in cancer-associated fibroblasts (CAFs) is linked to aggressive phenotypes and poor survival, and SDC2 overexpression in CAFs contributes to tumor growth. Our study provides a high-resolution GAC TME atlas and underscores potential targets for further investigation.


Asunto(s)
Adenocarcinoma , Fibroblastos Asociados al Cáncer , Lesiones Precancerosas , Neoplasias Gástricas , Humanos , Ecotipo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Adenocarcinoma/patología , Fibroblastos Asociados al Cáncer/patología , Lesiones Precancerosas/patología , Células del Estroma/patología , Microambiente Tumoral
13.
J Exp Clin Cancer Res ; 41(1): 257, 2022 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-35996148

RESUMEN

BACKGROUND: G protein-coupled receptor (GPCR) is the most targeted protein family by the FDA-approved drugs. GPCR-kinase 3 (GRK3) is critical for GPCR signaling. Our genomic analysis showed that GRK3 expression correlated with poor prognosis of gastric adenocarcinoma (GAC) patients. However, GRK3's functions and clinical utility in GAC progression and metastases are unknown. METHODS: We studied GRK3 expression in normal, primary, and metastatic GAC tissues. We identified a novel GRK3 inhibitor, LD2, through a chemical-library screen. Through genetic and pharmacologic modulations of GRK3, a series of functional and molecular studies were performed in vitro and in vivo. Impact of GRK3 on YAP1 and its targets was determined. RESULTS: GRK3 was overexpressed in GAC tissues compared to normal and was even higher in peritoneal metastases. Overexpression (OE) of GRK3 was significantly associated with shorter survival. Upregulation of GRK3 in GAC cells increased cell invasion, colony formation, and proportion of ALDH1+ cells, while its downregulation reduced these attributes. Further, LD2 potently and specifically inhibited GRK3, but not GRK2, a very similar kinase to GRK3. LD2 highly suppressed GAC cells' malignant phenotypes in vitro. Mechanistically, GRK3 upregulated YAP1 in GAC tissues and its transcriptional downstream targets: SOX9, Birc5, Cyr61 and CTGF. Knockdown (KD) YAP1 rescued the phenotypes of GRK3 OE in GAC cells. GRK3 OE significantly increased tumor growth but LD2 inhibited tumor growth in the PDX model and dramatically suppressed peritoneal metastases induced by GRK3 OE. CONCLUSIONS: GRK3, a poor prognosticator for survival, conferred aggressive phenotype. Genetic silencing of GRK3 or its inhibitor LD2 blunted GRK3-conferred malignant attributes, suggesting GRK3 as a novel therapeutic target in advanced GAC.


Asunto(s)
Adenocarcinoma , Neoplasias Peritoneales , Neoplasias Gástricas , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Peritoneales/genética , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo
14.
J Exp Clin Cancer Res ; 40(1): 207, 2021 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-34162421

RESUMEN

BACKGROUND: Gastric adenocarcinoma with peritoneal carcinomatosis (PC) is therapy resistant and leads to poor survival. To study PC in depth, there is an urgent need to develop representative PC-derived cell lines and metastatic models to study molecular mechanisms of PC and for preclinical screening of new therapies. METHODS: PC cell lines were developed from patient-derived PC cells. The tumorigenicity and metastatic potential were investigated by subcutaneously (PDXs) and orthotopically. Karyotyping, whole-exome sequencing, RNA-sequencing, and functional studies were performed to molecularly define the cell lines and compare genomic and phenotypic features of PDX and donor PC cells. RESULTS: We established three PC cell lines (GA0518, GA0804, and GA0825) and characterized them in vitro. The doubling times were 22, 39, and 37 h for GA0518, GA0804, and GA0825, respectively. Expression of cancer stem cell markers (CD44, ALDH1, CD133 and YAP1) and activation of oncogenes varied among the cell lines. All three PC cell lines formed PDXs. Interestingly, all three PC cell lines formed tumors in the patient derived orthotopic (PDO) model and GA0518 cell line consistently produced PC in mice. Moreover, PDXs recapitulated transcriptomic and phenotypic features of the donor PC cells. Finally, these cell lines were suitable for preclinical testing of chemotherapy and target agents in vitro and in vivo. CONCLUSION: We successfully established three patient-derived PC cell lines and an improved PDO model with high incidence of PC associated with malignant ascites. Thus, these cell lines and metastatic PDO model represent excellent resources for exploring metastatic mechanisms of PC in depth and for target drug screening and validation by interrogating GAC for translational studies.


Asunto(s)
Adenocarcinoma/patología , Perfilación de la Expresión Génica/métodos , Cariotipificación/métodos , Neoplasias Peritoneales/patología , Neoplasias Gástricas/patología , Adenocarcinoma/genética , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Ratones , Trasplante de Neoplasias , Neoplasias Peritoneales/genética , Análisis de Secuencia de ARN , Neoplasias Gástricas/genética , Secuenciación del Exoma
15.
Mol Cancer Res ; 18(3): 390-402, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31796534

RESUMEN

Despite established functions of PPARδ in lipid metabolism and tumorigenesis, the mechanisms underlying its role in gastric cancer are undefined. Here, we demonstrate that SOX9 was dramatically induced by stably expressing PPARδ and by its agonist GW501516 in human gastric cancer cell lines. PPARδ knockdown in patient-derived gastric cancer cells dramatically reduced SOX9 expression and transcriptional activity, with corresponding decreases in invasion and tumor sphere formation. Mechanistically, PPARδ induced SOX9 transcription through direct interaction with and activation of the Hippo coactivator YAP1. PPARδ-YAP1 interaction occurred via the C-terminal domain of YAP1, and both TEAD- and PPARE-binding sites were required for SOX9 induction. Notably, CRISPR/Cas9-mediated genetic ablation of YAP1 or SOX9 abolished PPARδ-mediated oncogenic functions. Finally, expression of PPARδ, YAP1, and SOX9 were significantly correlated with each other and with poor survival in a large cohort of human gastric cancer tissues. Thus, these findings elucidate a novel mechanism by which PPARδ promotes gastric tumorigenesis through interaction with YAP1 and highlights the PPARδ/YAP1/SOX9 axis as a novel therapeutic target in human gastric cancer. IMPLICATIONS: Our discovery of a new model supports a distinct paradigm for PPARδ and a crucial oncogenic function of PPARδ in gastric cancer through convergence on YAP1/TEAD signaling. Therefore, PPARδ/YAP1/SOX9 axis could be a novel therapeutic target that can be translated into clinics.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , PPAR delta/metabolismo , Factor de Transcripción SOX9/biosíntesis , Neoplasias Gástricas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Ratones , Ratones Desnudos , PPAR delta/genética , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Señalizadoras YAP
16.
PLoS One ; 15(2): e0228910, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32053639

RESUMEN

A fluorescence marker mOrange was inserted to the popular pLentiCrispr-V2 to create pLentiCrispr-V2-mOrange (V2mO) that contained both a puromycin selection and a fluorescent marker, making viral production and target transduction visible. Lentiviruses packaged with this plasmid and appropriate guide RNAs (gRNAs) successfully knocked out the genes RhoA, Gli1, and Gal3 in human gastric cancer cell lines. Cas9-gRNA editing efficiency could be estimated directly from Sanger electropherograms of short polymerase chain reaction products around the gRNA regions in Cas9-gRNA transduced cells. Single cloning of transduced target cell pools must be performed to establish stable knockout clones. Rescue of wildtype (RhoA and Gal3) and mutant (RhoA.Y42C) genes into knockout cells was successful only when cDNAs, where gRNAs bind, were modified by three nucleotides while the amino acid sequences remained unchanged. Stringent on-target CRISPR/Cas9 editing was observed in Gal3 gene, but not in RhoA gene since RhoA.Y42C already presented a nucleotide change in gRNA5 binding site. In summary, our improved strategy added these advantages: adding visual marker to the popular lentiviral system, monitoring lentiviral production and transduction efficiencies, cell-sorting Cas9+ cells in target cells by fluorescence-activated cell sorting, direct estimation of gene editing efficiency of target cell pools by short PCR electropherograms around gRNA binding sites, and successful rescue of wildtype and mutant genes in knockout cells, overcoming Cas9 editing by modifying cDNAs.


Asunto(s)
Técnicas de Inactivación de Genes/métodos , Ingeniería Genética/métodos , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Colorantes Fluorescentes , Edición Génica , Vectores Genéticos , Humanos , Lentivirus/genética , Plásmidos , ARN Guía de Kinetoplastida/genética
17.
Cancers (Basel) ; 12(10)2020 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-33076512

RESUMEN

Gastric adenocarcinoma (GAC) is inherently resistant or becomes resistant to therapy, leading to a poor prognosis. Mounting evidence suggests that lncRNAs can be used as predictive markers and therapeutic targets in the right context. In this study, we determined the role of lncRNA-PVT1 in GAC along with the value of inhibition of PVT1 using antisense oligos (ASOs). RNA scope in situ hybridization was used to analyze PVT1 expression in tumor tissue microarrays (TMAs) of GAC and paired normal tissues from 792 patients. Functional experiments, including colony formation and invasion assays, were performed to evaluate the effects of PVT1 ASO inhibition of PVT1 in vitro; patient-derived xenograft models were used to evaluate the anti-tumor effects of PVT1 ASOs in vivo. LncRNA-PVT1 was upregulated in GACs compared to the matched adjacent normal tissues in the TMA. LncRNA PVT1 expression was positively correlated with larger tumor size, deeper wall invasion, lymph node metastases, and short survival duration. Inhibition of PVT1 using PVT1 ASOs significantly suppressed tumor cell growth and invasion in vitro and in vivo. PVT1 expression was highly associated with poor prognosis in GAC patients and targeting PVT1 using PVT1 ASOs was effective at curtailing tumor cell growth in vitro and in vivo. Thus, PVT1 is a poor prognosticator as well as therapeutic target. Targeting PVT1 using PVT1 ASOs provides a novel therapeutic strategy for GAC.

18.
Mol Oncol ; 14(6): 1410-1426, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32175692

RESUMEN

Hippo/YAP1 signaling is a major regulator of organ size, cancer stemness, and aggressive phenotype. Thus, targeting YAP1 may provide a novel therapeutic strategy for tumors with high YAP1 expression in esophageal cancer (EC). Chromatin immunoprecipitation (ChiP) and quantitative ChiP-PCR were used to determine the regulation of the chromatin remodeling protein bromodomain-containing protein 4 (BRD4) on YAP1. The role of the bromodomain and extraterminal motif (BET) inhibitor JQ1, an established BRD4 inhibitor, on inhibition of YAP1 in EC cells was dissected using western blot, immunofluorescence, qPCR, and transient transfection. The antitumor activities of BET inhibitor were further examined by variety of functional assays, cell proliferation (MTS), tumorsphere, and ALDH1+ labeling in vitro and in vivo. Here, we show that BRD4 regulates YAP1 expression and transcription. ChiP assays revealed that BRD4 directly occupies YAP1 promoter and that JQ1 robustly blocks BRD4 binding to the YAP1 promoter. Consequently, JQ1 strongly suppresses constitutive or induced YAP1 expression and transcription in EC cells and YAP1/Tead downstream transcriptional activity. Intriguingly, radiation-resistant cells that acquire strong cancer stem cell traits and an aggressive phenotype can be effectively suppressed by JQ1 as assessed by cell proliferation, tumorsphere formation, and reduction in the ALDH1+ cells. Moreover, effects of JQ1 are synergistically amplified by the addition of docetaxel in vitro and in vivo. Our results demonstrate that BRD4 is a critical regulator of Hippo/YAP1 signaling and that BRD4 inhibitor JQ1 represents a new class of inhibitor of Hippo/YAP1 signaling, primarily targeting YAP1 high and therapy-resistant cancer cells enriched with cancer stem cell properties.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenocarcinoma/metabolismo , Azepinas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Neoplasias Esofágicas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Triazoles/farmacología , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Antineoplásicos/farmacología , Carcinogénesis/genética , Carcinogénesis/patología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Docetaxel/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Vía de Señalización Hippo , Humanos , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Regiones Promotoras Genéticas , Transcripción Genética/efectos de los fármacos , Proteínas Señalizadoras YAP
19.
Clin Cancer Res ; 25(7): 2264-2277, 2019 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-30563933

RESUMEN

PURPOSE: Esophageal cancer is a lethal disease that is often resistant to therapy. Alterations of YAP1 and CDK6 are frequent in esophageal cancer. Deregulation of both molecules may be responsible for therapy resistance. EXPERIMENTAL DESIGN: Expressions of YAP1 and CDK6 were examined in esophageal cancer cells and tissues using immunoblotting and immunohistochemistry. YAP1 expression was induced in esophageal cancer cells to examine YAP1-mediated CDK6 activation and its association with radiation resistance. Pharmacologic and genetic inhibitions of YAP1 and CDK6 were performed to dissect the mechanisms and assess the antitumor effects in vitro and in vivo. RESULTS: YAP1 expression was positively associated with CDK6 expression in resistant esophageal cancer tissues and cell lines. YAP1 overexpression upregulated CDK6 expression and transcription, and promoted radiation resistance, whereas treatment with the YAP1 inhibitor, CA3, strongly suppressed YAP1 and CDK6 overexpression, reduced Rb phosphorylation, as well as sensitized radiation-resistant/YAP1high esophageal cancer cells to irradiation. CDK4/6 inhibitor, LEE011, and knock down of CDK6 dramatically inhibited expression of YAP1 and sensitized resistant esophageal cancer cells to irradiation indicating a positive feed-forward regulation of YAP1 by CDK6. In addition, suppression of both the YAP1 and CDK6 pathways by the combination of CA3 and LEE011 significantly reduced esophageal cancer cell growth and cancer stem cell population (ALDH1 + and CD133 + ), sensitized cells to irradiation, and showed a strong antitumor effect in vivo against radiation-resistant esophageal cancer cells. CONCLUSIONS: Our results document that a positive crosstalk between the YAP1 and CDK6 pathways plays an important role in conferring radiation resistance to esophageal cancer cells. Targeting both YAP1 and CDK6 pathways could be a novel therapeutic strategy to overcome resistance in esophageal cancer.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Neoplasias Esofágicas/metabolismo , Tolerancia a Radiación , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Terapia Combinada , Quinasa 6 Dependiente de la Ciclina/genética , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/terapia , Expresión Génica , Técnicas de Inactivación de Genes , Genes Reporteros , Humanos , Inmunohistoquímica , Ratones , Modelos Biológicos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Tolerancia a Radiación/genética , Factores de Transcripción/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Señalizadoras YAP
20.
Mol Cancer Ther ; 17(2): 443-454, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29167315

RESUMEN

Mounting evidence suggests that the Hippo coactivator Yes-associated protein 1 (YAP1) is a major mediator of cancer stem cell (CSC) properties, tumor progression, and therapy resistance as well as often a terminal node of many oncogenic pathways. Thus, targeting YAP1 may be a novel therapeutic strategy for many types of tumors with high YAP1 expression, including esophageal adenocarcinoma. However, effective YAP1 inhibitors are currently lacking. Here, we identify a small molecule (CA3) that not only has remarkable inhibitory activity on YAP1/Tead transcriptional activity but also demonstrates strong inhibitory effects on esophageal adenocarcinoma cell growth especially on YAP1 high-expressing esophageal adenocarcinoma cells both in vitro and in vivo Remarkably, radiation-resistant cells acquire strong cancer stem cell (CSC) properties and aggressive phenotype, while CA3 can effectively suppress these phenotypes by inhibiting proliferation, inducing apoptosis, reducing tumor sphere formation, and reducing the fraction of ALDH1+ cells. Furthermore, CA3, combined with 5-FU, synergistically inhibits esophageal adenocarcinoma cell growth especially in YAP1 high esophageal adenocarcinoma cells. Taken together, these findings demonstrated that CA3 represents a new inhibitor of YAP1 and primarily targets YAP1 high and therapy-resistant esophageal adenocarcinoma cells endowed with CSC properties. Mol Cancer Ther; 17(2); 443-54. ©2017 AACR.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenocarcinoma/radioterapia , Neoplasias Esofágicas/radioterapia , Fosfoproteínas/metabolismo , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Neoplasias Esofágicas/patología , Humanos , Ratones , Ratones Transgénicos , Factores de Transcripción , Transfección , Proteínas Señalizadoras YAP
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