ABSTRACT
Cell signaling pathways and protein translation are crucial for understanding malignant transformation. 4E-BP1 and the eIF4F complex regulate cap-dependent translation. We investigated how 4E-BP1 and eIF4E phosphorylation status affects in vitro and in vivo cell proliferation in a breast cancer model. Cells from 2 breast carcinoma lines (MDA-MB 231 and MDA-MB 468) and human fibroblasts (IMR90 cells) were infected in vitro with a retrovirus carrying a wild-type 4E-BP1 or a mutant 4E-BP1 unable to hyperphosphorylate. Overexpression of the mutant 4E-BP1 induced a significant decrease in cell proliferation in IMR90 and MDA-MB 468 cells, but not in MDA-MB 231 cells. A correlation was observed between baseline-phosphorylated eIF4E (p-eIF4E) levels and sensitivity to 4E-BP1 transduction. By co-immunoprecipitation, p-eIF4E seemed to present lower affinity for 4E-BP1 than total eIF4E in MDA-MB 468 cells. After treatment with CGP57380, the MAP kinase-interacting kinase (MNK) inhibitor, downregulation of p-eIF4E levels was associated with an increase of E-cadherin and ß-catenin protein expression. These results provide evidence that 4E-BP1 transduction leads to a decrease in cell proliferation, and that high p-eIF4E levels may counteract the suppressor effect of 4E-BP1. We propose that high p-4E-BP1 and p-eIF4E levels are central factors in cell signaling and reflect the oncogenic potential of cell signaling pathways in breast cancer.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Breast Neoplasms/metabolism , Eukaryotic Initiation Factor-4E/metabolism , Phosphoproteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Breast Neoplasms/genetics , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Eukaryotic Initiation Factor-4E/genetics , Female , Gene Expression , Humans , Mice , Mice, Nude , Multiprotein Complexes/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Phosphoproteins/genetics , Phosphorylation , Protein Binding , Signal Transduction , Tumor Burden/genetics , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: The control of senescence and its biochemical pathways is a crucial factor for understanding cell transformation. In a large RNA interference screen, the RSK4 gene was found to be related to p53-dependent arrest. The purpose of the present study was to investigate the potential role of RSK4 as a tumor suppressor gene. EXPERIMENTAL DESIGN: RSK4 expression was determined by quantitative real-time PCR and immunoblot in 30 colon and 20 renal carcinomas, and in 7 colon adenomas. Two HCT116 colon carcinoma cell lines (p53 wt and p53 null), IMR90 human fibroblasts, and E1A-expressing IMR90 cells were infected with RSK4 cDNA and/or shRNA. RSK4 expression levels were analyzed in HCT116 p53 wt or p53 null and IMR90 after senescence induction by quantitative real-time PCR and Western blot. RESULTS: The RSK4 gene was down-regulated in 27 of 30 colon carcinomas (P < 0.001), 16 of 20 renal cell carcinomas (P < 0.01), and 6 of 7 colon adenomas (P < 0.01). In vitro overexpression of RSK4 induced cell arrest and senescence features in normal fibroblasts and malignant colon carcinoma cell lines. Interestingly, in these cell lines RSK4 mRNA levels were increased both in replicative and stress-induced senescence. Moreover, IMR90 partially immortalized by RSK4 shRNA and HCT116 with this short hairpin RNA were more resistant to cisplatin treatment. Finally, cells expressing E1A or Rb short interfering RNA were resistant to RSK4-mediated senescence. CONCLUSION: These results support the concept that RSK4 may be an important tumor suppressor gene by modulating senescence induction and contributing to cell proliferation control in colon carcinogenesis and renal cell carcinomas.
