Abstract
Relatively little is known about the molecular mechanisms of tumor promotion/progression in mammary carcinogenesis. Increased protein kinase C (PKC) activity is known to promote tumor formation in several tissues; however, its role in mammary carcinogenesis is not yet known. To determine if individual PKCs may selectively regulate properties of mammary tumor cells, we compared PKC isozyme levels in mammary tumor cell lines with low, moderate and high metastatic potential. All three cell lines expressed α, δ, ε and ζ PKCs; however, PKCδ levels were relatively increased in the highly metastatic cells. To determine if increased PKCδ could contribute to promotion/progression, we overexpressed PKCδ in the low and moderately metastatic cell lines. PKCδ overexpression had no significant effect on growth of adherent cells, but significantly increased anchorage-independent growth. Conversely, expressing the regulatory domain of PKCδ (RDδ), a putative PKCδ inhibitory fragment, inhibited anchorage-independent growth. The efficacy of RDδ as a PKCδ inhibitor was demonstrated by showing that RDδ selectively interfered with PKCδ subcellular location and significantly interfered with phosphorylation of the PKC cytoskeletal substrate, adducin. PKC-dependent phosphorylation of cytoskeletal substrate proteins, such as adducin, provides a mechanistic link between increased PKCδ activity and phenotypic changes in cytoskeletal-dependent processes such as migration and attachment, two processes that are relevant to metastatic potential. The reciprocal growth effects of expressing PKCδ and RDδ as gain and loss of function constructs, respectively, provide strong evidence that PKCδ regulates processes important for anchorage-independent growth in these mammary tumor cells.
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Abbreviations
- DAG:
-
diacylglycerol
- HA:
-
hemagglutinin
- MT:
-
mammary tumor
- MTT:
-
4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide
- PBS:
-
phosphate-buffered saline
- PKC:
-
protein kinase C
- RD:
-
regulatory domain
- wt:
-
wild type
- Zn:
-
zinc chloride
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Acknowledgements
This work was supported by US Army Medical Research and Materiel Command Grant #DAMD17-94-J4312 (S Jaken), #DAMD17-96-1-6152 (DR Welch) and NIH grant CA #71604 (S Jaken). We would like to thank Katrina Ramsay, Breandan Kennedy and Betty Mousseau for their individual contributions to the evolution of the DNA constructs used in this study. Marina LaDuke and Alice Vera assisted in the preparation of figures contained within this manuscript.
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Kiley, S., Clark, K., Duddy, S. et al. Increased protein kinase Cδ in mammary tumor cells: relationship to transformation and metastatic progression. Oncogene 18, 6748–6757 (1999). https://doi.org/10.1038/sj.onc.1203101
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DOI: https://doi.org/10.1038/sj.onc.1203101
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