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PTEN can inhibit in vitro organotypic and in vivo orthotopic invasion of human bladder cancer cells even in the absence of its lipid phosphatase activity

Oncogene volume 23, pages 6788–6797 (2004)Cite this article

Abstract

Recent studies have found a higher frequency of the PTEN tumor-suppressor gene alterations in invasive bladder carcinoma than in superficial disease, suggesting that PTEN is important in this process. A role of PTEN in bladder cancer invasion is further suggested by the fact that PTEN is a regulator of cell motility, a necessary component of tumor invasion. However, it is unknown whether PTEN is mechanistically involved in ‘in vivo’ tumor invasion or merely an epiphenomenon and, if the former is true, whether this process is dependent on its protein or lipid phosphatase activities. To address these issues, we stably transfected several commonly used human bladder cancer cell lines with known invasive phenotypes with either wild-type PTEN constructs or those deficient in the lipid phosphatase (G129E) or both protein and lipid phosphatase (G129R) activities. Here we show that chemotaxis was inhibited by both the wild-type and G129E mutant of PTEN but not by G129R-transfected cells. Using a novel organotypic in vitro invasion assay, we evaluated the impact of wild-type and mutant PTEN transgene expression on the invasive ability of T24T, a human bladder cancer cell line with a functionally impaired PTEN. Results indicate that the G129E mutant blocks invasion as efficiently as wild-type PTEN transfection. In contrast to the wild-type gene, this mutant has no effect on cell clonogenicity in agar. To further establish the role of PTEN in tumor invasion, we evaluated vector- and PTEN-transfected T24T cells in an orthotopic in vivo assay that faithfully reproduces human disease. Microscopic examination of murine bladders at the completion of this experiment parallels the results obtained with the organotypic assay. Our results are the first demonstration: (1) that the inhibitory effects of PTEN on cell motility translate into suppression of in vivo invasion; (2) that PTEN can inhibit tumor invasion even in the absence of its lipid phosphatase activity; (3) how organotypic in vitro approaches can be used as surrogates of in vivo invasion allowing rapid dissection of molecular processes leading to this phenotype while reducing the number of animals used in research.

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Acknowledgements

We thank Dr A Klippel for the generous gift of dominant-negative PI3K p85 subunit, Drs Frank Furnari and Webster Cavenee for the PTEN constructs and PBABE vector used for generation of both transient and stable transfections and Kathryn M Gulding for expert technical assistance. This work was supported by an NIH training grant DK007766 to JJG and NIH R01CA085329 awards to DT JJG is an American Foundation for Urologic Disease (AFUD) scholar.

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  1. John J Gildea and Mikael Herlevsen: Contributed equally to this work

Authors and Affiliations

  1. Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Box 422, Charlottesville, 22908, VA, USA

    John J Gildea, Mikael Herlevsen, Michael A Harding, Kathryn M Gulding & Dan Theodorescu

  2. Department of Pathology, University of Virginia Health Sciences Center, Box 572, Charlottesville, 22908, VA, USA

    Christopher A Moskaluk & Henry F Frierson

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  1. John J Gildea
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Correspondence to Dan Theodorescu.

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Gildea, J., Herlevsen, M., Harding, M. et al. PTEN can inhibit in vitro organotypic and in vivo orthotopic invasion of human bladder cancer cells even in the absence of its lipid phosphatase activity. Oncogene 23, 6788–6797 (2004). https://doi.org/10.1038/sj.onc.1207599

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