Reduced expression of Wnt-1 and E-cadherin, and diminished beta-catenin stability in MCF-7 breast cancer cells that overexpress protein kinase C-alpha.
MCF-7 breast cancer cells stably overexpressing protein kinase C-alpha (MCF-7-PKC-alpha cells) exhibit reduced cell-cell adhesion and increased tumorigenicity in nude mice. We investigated the possibility that alterations in E-cadherin and catenins contribute to the unique phenotype of MCF-7-PKC-alpha cells. Northern and Western blotting indicated that MCF-7-PKC-alpha cells express abnormally low amounts of plakoglobin mRNA and protein, and undetectable levels of E-cadherin mRNA and protein. In contrast, even though MCF-7-PKC-alpha cells express low levels of beta-catenin mRNA, they express undetectable levels of beta-catenin protein, suggesting that post-transcriptional events further diminish beta-catenin expression in these cells. Pulse-labeling of the cells with [35S]methionine showed that the half-life of beta-catenin is less than 15 min in MCF-7-PKC-alpha cells, compared to over 2 h in MCF-7-Vector cells [MCF-7 cells transfected with pSV2M(2)6 vector only]. Incubation with LiCl to inactivate glycogen synthase kinase-3 (GSK-3) significantly prolonged the half-life of beta-catenin in MCF-7-PKC-alpha cells, suggesting that the GSK-3-dependent degradation of beta-catenin contributes to beta-catenin instability in these cells. Northern and Western blotting indicated that Wnt-1, which also inhibits GSK-3 activity, is expressed by MCF-7-Vector cells, but not by MCF-7-PKC-alpha cells. Transfection of (S37A)beta-catenin, which is resistant to GSK-3-dependent degradation, stimulated TCF/LEF-dependent luciferase expression from the pTOPFLASH reporter plasmid by 753-fold in MCF-7-PKC-alpha cells, and by 268-fold in MCF-7-Vector cells. Inactivation of GSK-3 by LiCl stimulated luciferase expression from the pTOPFLASH reporter plasmid by 12.4-fold in MCF-7-PKC-alpha cells, and by 4.8-fold in MCF-7-Vector cells. These results suggest that degradation of beta-catenin by GSK-3 contributes to beta-catenin instability in MCF-7-PKC-alpha cells, diminishing the ability of -catenin to act as a transcriptional co-activator. Reduced Wnt-1 expression by MCF-7-PKC-alpha cells may promote beta-catenin degradation by enhancing GSK-3 activity. Loss of beta-catenin-dependent cell-cell adhesion and transcription may contribute to the aggressive phenotype of MCF-7-PKC-alpha cells.
Williams
C L
CL
Laboratory of Molecular Pharmacology, Guthrie Research Institute, Sayre, PA 18840, USA.
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JD
D000818:Animals; D015152:Blotting, Northern; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005260:Female; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D011493:Protein Kinase C / Q000378:metabolism; D051571:Protein Kinase C-alpha; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D015534:Trans-Activators; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism; D051153:Wnt Proteins; D051155:Wnt1 Protein; D029961:Zebrafish Proteins; D051176:beta Catenin
C495270:CTNNB1 protein, human; D015820:Cadherins; D003598:Cytoskeletal Proteins; D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; C495247:WNT1 protein, human; D051153:Wnt Proteins; D051155:Wnt1 Protein; D029961:Zebrafish Proteins; D051176:beta Catenin; D008156:Luciferases; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha