Hunk is required for HER2/neu-induced mammary tumorigenesis
Elizabeth S. Yeh, Thomas W. Yang, Jason J. Jung, Heather P. Gardner, Robert D. Cardiff, Lewis A. Chodosh
Elizabeth S. Yeh, Thomas W. Yang, Jason J. Jung, Heather P. Gardner, Robert D. Cardiff, Lewis A. Chodosh
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Research Article Oncology

Hunk is required for HER2/neu-induced mammary tumorigenesis

Abstract

Understanding the molecular pathways that contribute to the aggressive behavior of human cancers is a critical research priority. The SNF1/AMPK-related protein kinase Hunk is overexpressed in aggressive subsets of human breast, ovarian, and colon cancers. Analysis of Hunk–/– mice revealed that this kinase is required for metastasis of c-myc–induced mammary tumors but not c-myc–induced primary tumor formation. Similar to c-myc, amplification of the proto-oncogene HER2/neu occurs in 10%–30% of breast cancers and is associated with aggressive tumor behavior. By crossing Hunk–/– mice with transgenic mouse models for HER2/neu-induced mammary tumorigenesis, we report that Hunk is required for primary tumor formation induced by HER2/neu. Knockdown and reconstitution experiments in mouse and human breast cancer cell lines demonstrated that Hunk is required for maintenance of the tumorigenic phenotype in HER2/neu-transformed cells. This requirement is kinase dependent and resulted from the ability of Hunk to suppress apoptosis in association with downregulation of the tumor suppressor p27kip1. Additionally, we find that Hunk is rapidly upregulated following HER2/neu activation in vivo and in vitro. These findings provide what we believe is the first evidence for a role for Hunk in primary tumorigenesis and cell survival and identify this kinase as an essential effector of the HER2/neu oncogenic pathway.

Authors

Elizabeth S. Yeh, Thomas W. Yang, Jason J. Jung, Heather P. Gardner, Robert D. Cardiff, Lewis A. Chodosh

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Figure 2

Inhibition of HER2/neu activity downregulates Hunk.

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Inhibition of HER2/neu activity downregulates Hunk. (A) QRT-PCR analysis...
(A) QRT-PCR analysis of neu and Hunk in MTB/TAN cells at 0, 24, and 96 hours after doxycycline removal from growth medium. (B) Western blot analysis for Hunk in MCF10A cells transduced with wild-type HER2 or a control vector. (C) Western blot analysis of Hunk, phospho-Akt, and phospho-ERK1/2 in BT474 cells treated with DMSO (vehicle) or 100 nM or 200 nM lapatinib for 24 hours. (D) QRT-PCR analysis of Hunk mRNA levels in BT474 cells treated with DMSO (vehicle) or 100 nM or 200 nM lapatinib for 24 hours. (E and F) QRT-PCR (E) and Western blot (F) analysis of Hunk levels in BT474 cells treated with DMSO (vehicle) or 100 nM lapatinib for 0, 6, 12, or 24 hours. (G) Western blot analysis of Hunk in SMF cells treated with DMSO (vehicle) or 20 nM LY249002 for 1 or 6 hours. (H) Western blot analysis of Hunk in SMF cells treated with DMSO (vehicle) or 1 mM PD184352 for 1, 3, or 6 hours. (I) Western blot analysis of Hunk protein levels in BT474 cells stimulated with EGF after 24 hours of serum withdrawal. Cells were stimulated with 5 μg/ml EGF for 24, 48, or 96 hours. (J) Western blot analysis of EGFR, HER3, and HER4 bound to immunoprecipitated HER2. (K) Western blot analysis of BT474 cells treated with DMSO (vehicle), 100 nM lapatinib, 100 nM gefitinib, or ATM inhibitor (negative control) for 24 hours.