ABSTRACT
The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21(/Cip) and p27(/Kip1). Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.
Subject(s)
Cell Cycle/genetics , Cellular Senescence/genetics , Proteasome Endopeptidase Complex/deficiency , Trans-Activators/deficiency , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Survival/genetics , Cyclin B1/genetics , Cyclin B1/metabolism , Cyclin D1/genetics , Cyclin D1/metabolism , Cyclin-Dependent Kinase Inhibitor Proteins/genetics , Cyclin-Dependent Kinase Inhibitor Proteins/metabolism , DNA/analysis , Epithelial Cells/cytology , Epithelial Cells/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , G1 Phase/genetics , Gene Expression/genetics , HeLa Cells , Humans , Phosphorylation/genetics , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , RNA, Small Interfering/genetics , Resting Phase, Cell Cycle/genetics , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , Sulfotransferases/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transfection , Ubiquitinated Proteins/metabolism , Up-Regulation/genetics , beta-Galactosidase/metabolism , cdc25 Phosphatases/genetics , cdc25 Phosphatases/metabolismABSTRACT
Genomes of human cancer cells are characterized by numerous chromosomal aberrations of uncertain pathogenetic significance. Here, in an inducible mouse model of melanoma, we characterized metastatic variants with an acquired focal chromosomal amplification that corresponds to a much larger amplification in human metastatic melanomas. Further analyses identified Nedd9, an adaptor protein related to p130CAS, as the only gene within the minimal common region that exhibited amplification-associated overexpression. A series of functional, biochemical, and clinical studies established NEDD9 as a bona fide melanoma metastasis gene. NEDD9 enhanced invasion in vitro and metastasis in vivo of both normal and transformed melanocytes, functionally interacted with focal adhesion kinase and modulated focal contact formation, and exhibited frequent robust overexpression in human metastatic melanoma relative to primary melanoma. Thus, comparative oncogenomics has enabled the identification and facilitated the validation of a highly relevant cancer gene governing metastatic potential in human melanoma.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Melanoma/genetics , Melanoma/secondary , Oncogenes , Phosphoproteins/genetics , Animals , Base Sequence , DNA, Neoplasm/genetics , Female , Focal Adhesion Kinase 1/physiology , Gene Amplification , Gene Expression , Genomics , Humans , In Vitro Techniques , Melanocytes/metabolism , Melanocytes/pathology , Melanoma/pathology , Melanoma, Experimental/genetics , Melanoma, Experimental/pathology , Melanoma, Experimental/secondary , Mice , Mice, Knockout , Mice, SCID , Neoplasm Invasiveness , RNA Interference , Species SpecificityABSTRACT
The pancreatic adenocarcinoma genome harbors multiple amplifications and deletions, pointing to the existence of numerous oncogenes and tumor suppressor genes driving the genesis and progression of this lethal cancer. Here, array comparative genomic hybridization on a cDNA microarray platform and informatics tools have been used to define the copy number alterations in a panel of 24 pancreatic adenocarcinoma cell lines and 13 primary tumor specimens. This high-resolution genomic analysis has identified all known regional gains and losses as well as many previously uncharacterized highly recurrent copy number alterations. A systematic prioritization scheme has selected 64 focal minimal common regions (MCRs) of recurrent copy number change. These MCRs possess a median size of 2.7 megabases (Mb), with 21 (33%) MCRs spanning 1 Mb or less (median of 0.33 Mb) and possessing an average of 15 annotated genes. Furthermore, complementary expression profile analysis of a significant fraction of the genes residing within these 64 prioritized MCRs has enabled the identification of a subset of candidates with statistically significant association between gene dosage and mRNA expression. Thus, the integration of DNA and RNA profiles provides a highly productive entry point for the discovery of genes involved in the pathogenesis of pancreatic adenocarcinoma.
