The gene for Pregnancy Up-regulated Non-ubiquitous Calmodulin Kinase (Pnck), a novel calmodulin kinase, is expressed in roughly one-third of human breast tumors, but not in adjoining normal tissues. Pnck alters EGFR stability and function, prompting this study to determine if Pnck expression has implications for HER-2 function and HER-2-directed therapy. The frequency of Pnck expression in HER-2-amplified breast cancer was examined by immunohistochemistry, and the impact of Pnck expression in the presence of HER-2 amplification on cancer cell proliferation, clonogenicity, cell-cycle progression, and Trastuzumab sensitivity was examined in vitro by transfection of cells with Pnck. Cell signaling was probed by Western blot analysis and shRNA-mediated PTEN knockdown. Over 30 % of HER-2 amplified tumors were found to express Pnck. Expression of Pnck in SkBr3 cells resulted in increased proliferation, clonal growth, cell-cycle progression, and Trastuzumab resistance. Pnck expression increases Hsp27 expression, Trastuzumab partial agonist activity on HER-2 Y1248 phosphorylation, and suppressed extracellular signal-regulated kinase (ERK1/2) activity. Knockdown of endogenous PTEN upregulated ERK1/2 activity, inhibited cellular proliferation, and partially sensitized Pnck/SKBr3 cells to Trastuzumab treatment. Increased proliferation of the Pnck/SKBr3 cells was observed following expression of protein phosphatase active and lipid phosphatase dead PTEN mutant but not the total phosphatase dead PTEN mutant. Co-overexpression of HER-2 and Pnck results in enhanced tumor cell proliferation and Trastuzumab resistance that is paradoxically dependent on PTEN protein phosphatase activity. This suggests that Pnck may be a marker of Trastuzumab resistance and possibly a therapeutic target.
Bibliographical noteFunding Information:
We sincerely thank Genentech, Inc. (currently Roche, Inc.) for the kind gift of Trastuzumab (Herceptin). This work is supported by financial assistance from US Department of Defense BCRP Concept award (BC103388), an award from Susan G. Komen Foundation for Cure (BCTR0707114), an American Cancer Society Institutional Research Grant (IRG-97-152-16), a grant from Nina Hyde Breast Cancer Research Center, a Cancer Center Support Grant developmental fund award (CCSG DFA) (all to TBD). Views expressed in this publication are solely authors’ view and US Department of Defense and National Institutes of Health may not necessarily agree with these views. We sincerely thank assistance from Lombardi Cancer Center Shared Resources which are funded by Cancer Center Support Grant 5P30CA051008-17 from National Institutes of Health. All authors have read and approved the manuscript.
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