ABSTRACT
Minichromosome maintenance (MCM) proteins 2-7 are important in DNA replication licensing. Functional roles beyond licensing are speculated. In addition, significances in medulloblastoma (MB) remain unclear. In this study, we showed the frequent deregulation of MCM2 and MCM3 expression in 7 MB cell lines and 31 clinical samples. Moreover, DAOY and ONS76 and the clinical samples expressed elevated MCM7 transcripts with genomic gain of the gene. Immunopositivity restricted to tumor cells was found in 41, 37 and 53 out of 73 MB cases for MCM2, MCM3 and MCM7, respectively. High-MCM3 expression was associated with poor prognosis. Knockdowns of these MCMs significantly inhibited anchorage-dependent and -independent MB cell growth. The inhibition of MCM3 expression by small interfering RNA knockdown was related to G1 arrest with reduced cyclin A expression, whereas the MCM2- and MCM7-knocked-down cells arrested at G2/M with increased cyclin A expression. Interestingly, we demonstrated the links of these MCMs with cell migration and invasion using wound-healing and Transwell migration/invasion assays. Exogenous overexpression of MCM2, MCM3 and MCM7 increased anchorage-independent cell growth, and also cell migration and invasion capabilities in MB cells. The knockdown reduced the number of filopodial cells and the cells with intense stress fibers by blocking cdc42 and Rho activation. Taken together, deregulation of MCM2, MCM3 and MCM7 expression might be involved in MB tumorigenesis and we revealed undefined roles of these MCMs in control of MB cell migration and invasion.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Movement/genetics , DNA-Binding Proteins/metabolism , Medulloblastoma/metabolism , Nuclear Proteins/metabolism , Biomarkers, Tumor/analysis , Cell Cycle Proteins/genetics , Cell Separation , Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/metabolism , Cerebellar Neoplasms/pathology , Comparative Genomic Hybridization , DNA-Binding Proteins/genetics , Flow Cytometry , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Kaplan-Meier Estimate , Medulloblastoma/genetics , Medulloblastoma/pathology , Minichromosome Maintenance Complex Component 2 , Minichromosome Maintenance Complex Component 3 , Minichromosome Maintenance Complex Component 7 , Neoplasm Invasiveness/genetics , Nuclear Proteins/genetics , Oligonucleotide Array Sequence Analysis , Prognosis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
G-protein-coupled receptor-30 (GPR30) shows estrogen-binding affinity and mediates non-genomic signaling of estrogen to regulate cell growth. We here showed for the first time, in contrast to the reported promoting action of GPR30 on the growth of breast and ovarian cancer cells, that activation of GPR30 by the receptor-specific, non-estrogenic ligand G-1 inhibited the growth of androgen-dependent and androgen-independent prostate cancer (PCa) cells in vitro and PC-3 xenografts in vivo. However, G-1 elicited no growth or histological changes in the prostates of intact mice and did not inhibit growth in quiescent BPH-1, an immortalized benign prostatic epithelial cell line. Treatment of PC-3 cells with G-1 induced cell-cycle arrest at the G(2) phase and reduced the expression of G(2)-checkpoint regulators (cyclin-A2, cyclin-B1, cdc25c, and cdc2) and phosphorylation of their common transcriptional regulator NF-YA in PC-3 cells. With extensive use of siRNA-knockdown experiments and the MEK inhibitor PD98059 in this study, we dissected the mechanism underlying G-1-induced inhibition of PC-3 cell growth, which was mediated through GPR30, followed by sustained activation of Erk1/2 and a c-jun/c-fos-dependent upregulation of p21, resulting in the arrest of PC-3 growth at the G(2) phase. The discovery of this signaling pathway lays the foundation for future development of GPR30-based therapies for PCa.
Subject(s)
Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , G2 Phase/drug effects , Prostatic Neoplasms/drug therapy , Proto-Oncogene Proteins/metabolism , Receptors, G-Protein-Coupled/agonists , Animals , Antineoplastic Agents/pharmacology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Nucleus/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclopentanes/pharmacology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Flavonoids/pharmacology , Gene Expression/drug effects , Gene Expression/genetics , Humans , Male , Mice , Mice, Nude , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation/drug effects , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Quinolines/pharmacology , RNA, Small Interfering/genetics , Receptors, Estrogen/antagonists & inhibitors , Receptors, G-Protein-Coupled/genetics , Signal Transduction/drug effects , Signal Transduction/physiology , Xenograft Model Antitumor AssaysABSTRACT
Epidemiological studies suggested that ovulation was associated with ovarian carcinogenesis. Follicle-stimulating hormone (FSH) played an important role in follicular development and was recently found to affect growth of ovarian epithelial cells. Single nucleotide polymorphisms (SNPs) Thr307Ala and Asn680Ser were two non-synonymous variations in the coding region of the FSH receptor (FSHR) gene. This hitherto first case-control study investigating the association between these two FSHR SNPs and the risk of ovarian cancer involved 202 histopathologically confirmed ovarian cancer patients and 266 age-matched cancer-free control subjects using restriction fragment length polymorphism assay and direct sequencing. Our results demonstrated that the 307Ala and 680Ser carriers were associated with significantly increased risk of developing serous and mucinous types of ovarian cancers (P < 0.0005, OR = 2.60, 95% CI = 1.56-4.34; and P < 0.0005, OR = 2.89, 95% CI = 1.73-4.84, adjusted for age, respectively) but not endometrioid and clear cell types. The two SNPs were found to be in modest linkage disequilibrium, D' = 0.804 and 0.701, r2 = 0.581 and 0.406 for the cancer and control groups, respectively. The major haplotype of 307Ala-680Ser was also associated with higher cancer risk (P = 0.033, OR = 1.39, 95% CI = 1.03-1.88), especially for the serous and mucinous carcinomas (P = 0.001, OR = 1.82, 95% CI = 1.27-2.60). Our results suggested that the two FSHR SNPs might affect the susceptibility of women to specific subtypes of ovarian cancer. Different types of ovarian cancer might adopt distinct carcinogenetic pathways. Such understanding may be important in selecting patients for ovulation induction therapy.
