ABSTRACT
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.
Subject(s)
Adenocarcinoma , Peritoneal Neoplasms , Stomach Neoplasms , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Humans , Peritoneal Neoplasms/genetics , Stomach Neoplasms/drug therapy , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolismABSTRACT
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.
Subject(s)
Adenocarcinoma/pathology , Gene Expression Profiling/methods , Karyotyping/methods , Peritoneal Neoplasms/pathology , Stomach Neoplasms/pathology , Adenocarcinoma/genetics , Animals , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Mice , Neoplasm Transplantation , Peritoneal Neoplasms/genetics , Sequence Analysis, RNA , Stomach Neoplasms/genetics , Exome SequencingABSTRACT
BACKGROUND: Approximately one third of needle biopsies that are performed to rule out malignancy of indeterminate pulmonary nodules detected radiologically during lung cancer screening are negative, thus exposing cancer-free patients to risks of pneumothorax, bleeding, and infection. A noninvasive confirmatory tool (eg, liquid biopsy) is urgently needed in the lung cancer diagnosis setting to stratify patients who should receive biopsy versus those who should be monitored. METHODS: A novel antigen-independent, 4-color fluorescence in situ hybridization (FISH)-based method was developed to detect circulating tumor cells (CTCs) with abnormalities in gene copy numbers in mononuclear cell-enriched peripheral blood samples from patients with (n = 107) and without (n = 100) lung cancer. RESULTS: Identification of CTCs using FISH probes at 10q22.3/CEP10 and 3p22.1/3q29 detected lung cancer cases with 94.2% accuracy, 89% sensitivity, and 100% specificity compared with biopsy. CONCLUSION: The high accuracy of this liquid biopsy method suggests that it may be used as a noninvasive decision tool to reduce the frequency of unnecessary needle biopsy in patients with benign pulmonary lesions.
Subject(s)
Lung Diseases/diagnosis , Lung Neoplasms/diagnosis , Neoplastic Cells, Circulating , Tomography, X-Ray Computed/methods , A549 Cells , Aged , Aneuploidy , Diagnosis, Differential , Female , Humans , In Situ Hybridization, Fluorescence/methods , Liquid Biopsy , Lung Diseases/diagnostic imaging , Lung Diseases/genetics , Lung Neoplasms/diagnostic imaging , Lung Neoplasms/genetics , Male , Middle Aged , Neoplasm Staging , Sensitivity and SpecificityABSTRACT
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.