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Int6 regulates both proteasomal degradation and translation initiation and is critical for proper formation of acini by human mammary epithelium

Oncogene volume 30, pages 724–736 (2011)Cite this article

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Abstract

INT6/EIF3E has been implicated in breast tumorigenesis, but its functional activities remain poorly defined. We found that, repressing INT6 expression induced transformed properties in normal human mammary epithelium (MCF10A); in contrast, Int6 silencing induced apoptosis in HeLa cells. As in fission yeast, Int6 in human cells was required for assembly of active proteasomes. A reverse-phase protein array screen identified SRC3/AIB1 as one oncoprotein the level and stability of which increased when Int6 was silenced in MCF10A cells. Our data further show that Int6 binds SRC3 and its ubiquitin ligase Fbw7, thus perhaps mediating the interaction between SRC3–Fbw7 and proteasomes. Consistent with this, Int6 silencing did not increase SRC3 levels in HeLa cells, which have low Fbw7 levels. It is surprising that, however, polyubiquitylated proteins do not accumulate or may even decrease in Int6-silenced cells that contain defective proteasomes. Considering that decreased ubiquitin might explain this observation and that Int6 might control ubiquitin levels in its role as a subunit of eIF3 (eukaryote translation initiation factor 3), we found that silencing Int6 reduced monoubiquitin protein levels, which correlated with a shift of ubiquitin mRNAs from larger polysomes to non-translating ribosomes. In contrast, levels of many housekeeping proteins did not change. This apparent reduction in the translation of ubiquitin genes correlated with a modest reduction in protein synthesis rate and formation of large polysomes. To further determine whether Int6 can selectively control translation, we analyzed translation of different 5′-untranslated region reporters and found that indeed, loss of Int6 had differential effects on these reporters. Together the data suggest that Int6 depletion blocks ubiquitin-dependent proteolysis by decreasing both ubiquitin levels and the assembly of functional proteasome machinery, leading to accumulation of oncoproteins, such as SRC3 that can transform mammary epithelium. Our data also raise the possibility that Int6 can further fine-tune protein levels by selectively controlling translation of specific mRNAs.

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Acknowledgements

We thank John Hershey for providing eIF3 antibodies, and members of the Adrian Lee and Steffi Oesterreich labs for the help with tissue culture. We thank Carlos Rivera for technical assistance in the translation study and Steen Pedersen for assistance in the use of the fluorometer, Chris Threetor, Mike Cubbage and Xinrong Fu for the use of a flow cytometer in the Flow Cytometry core lab at Texas Children's Cancer Center, Michael Lewis for confocal microscopy, Gary Chamness for reading the manuscript, Anna Tsimelzon and Susan Hilsenbeck for database analyses, Jan Sap for assistance in Int6 expression studies, Yiling Lu for assistance in the RRPA screen, members of Robert Callahan's group for discussion and David Lonard and Bert O’Malley in the help of SRC3 study. We are particularly grateful to Sizhen Gao, Joan Brugge and Sean McGuire for sharing unpublished results and comments for the paper. ECC is supported by grants from the NIH (CA90464, CA107187 and P50CA58183), JS by fellowships from the Susan G. Komen Foundation (PDF0402733) and Expedition Inspiration Fund for Breast Cancer Research, RS by grants from NIH, P50CA058183 and SU2C (Stand Up 2 Cancer) and REL by NIH grants (AI50237 and GM59803).

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology and Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA

    J Suo & E C Chang

  2. Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA

    S J Snider & R E Lloyd

  3. Department of Systems Biology, M D Anderson Cancer Center, The University of Texas, Houston, TX, USA

    G B Mills

  4. Division of Biostatistics and Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA

    C J Creighton

  5. Department of Medicine and Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA

    A C Chen & R Schiff

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  1. J Suo
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Correspondence to E C Chang.

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Suo, J., Snider, S., Mills, G. et al. Int6 regulates both proteasomal degradation and translation initiation and is critical for proper formation of acini by human mammary epithelium. Oncogene 30, 724–736 (2011). https://doi.org/10.1038/onc.2010.445

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