Hunk is required for HER2/neu-induced mammary tumorigenesis
Elizabeth S. Yeh, … , Robert D. Cardiff, Lewis A. Chodosh
Elizabeth S. Yeh, … , Robert D. Cardiff, Lewis A. Chodosh
Published March 1, 2011; First published February 14, 2011
Citation Information: J Clin Invest. 2011;121(3):866-879. https://doi.org/10.1172/JCI42928.
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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 1

HER2/neu upregulates Hunk.

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HER2/neu upregulates Hunk. (A) Northern blot analysis of Hunk transcript...
(A) Northern blot analysis of Hunk transcript levels in non-transformed mammary cell lines (non-T) and transformed cell lines isolated from tumors derived from MMTV-Fgf3/int, MMTV–c-Myc, MMTV-neu, and MMTV–H-Ras mice. (Lanes 1: 3T3; 2: NMuMG; 3: HC11; 4: CL-S1; 5: HBI2; 6: 1128; 7: 8Ma1a; 8: MBp6; 9: M1011; 10: M158; 11: 16MB9a; 12: SMF; 13: NAF; 14: NF639; 15: NF11005; 16: NK-2; 17: AC816; 18: AC711; 19: AC236.) (B) Hunk levels as determined by QRT-PCR in non-transformed mammary cell lines HC11 and NMuMG and HER2/neu-transformed cell lines SMF and NAF. (C) Western blot analysis of Hunk and HER2/neu protein levels in HC11, NMuMG, SMF, and NAF cells. (D) Western blot analysis of Hunk protein levels in HC11 cells stably expressing an empty vector control (HC11-control), an activated allele of neu (HC11-neu), or SMF cells. Hunk was immunoprecipitated from 1 mg total protein of each individual cell type. (E) Hunk transcript levels as determined by QRT-PCR in mammary glands isolated from MTB/TAN mice induced with doxycycline for 96 hours, represented as the average level of Hunk expression in individual mammary glands (n = 3). P < 0.01. Data represent mean ± SEM. (F and G) QRT-PCR analysis of neu (F) and Hunk (G) in mammary glands (MG) isolated from wild-type FVB mice or in architecturally normal mammary glands from 10-week-old MMTV-neu mice, hyperplastic non–tumor-bearing (NTB) glands from MMTV-neu mice, or tumors from MMTV-neu mice.