Abstract P5-04-06: Loss of the E3 ubiquitin ligase HACE1 plays a critical role in transformation of mammary cells and clinical progression of human breast cancer via accumulation of active Rac1

Abstract

Abstract Invasive ductal breast cancer arises when the immediate precursor, ductal carcinoma in situ (DCIS), breaches the basement membrane of the ductal lumen and invades into the surrounding tissue. Although this transition has been implicated as a crucial step in the progression of breast cancer, the specific alterations responsible for this transition have yet to be identified. HER2/neu, a member of the epidermal growth family receptor (EGFR) family, is frequently overexpressed in non-invasive lesions (50-60%) but is significantly less common in invasive breast cancer (20-30%). This suggests that while HER2/neu may be partially transformative, additional alterations are required for mammary epithelial cells to achieve full malignant transformation. To identify novel genes capable of cooperating with HER2, we performed an insertional mutagenesis screen for genes whose alteration induces anchorage-independent growth on primary mouse mammary epithelial cells isolated from MMTV-neu transgenic mice. We identified HECT domain and ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) as a putative breast tumor suppressor gene whose loss contributes to the transformative process. Loss of HACE1 expression is commonly seen in publicly available breast cancer patient data sets as well as in established breast cancer cell lines supporting the role of HACE1 as a breast cancer tumor suppressor gene. Knockdown of HACE1 in HER2 overexpressing human mammary epithelial cells (HMECs) enhanced colony formation over HER2 overexpressing HMEC controls cells. Moreover, knockdown of HACE1 alone was sufficient to allow anchorage-independent growth in soft agar in the HMECs while the overexpression of HACE1 in breast cancer cell lines diminishes clonogenic capacity in soft agar. We confirmed recent studies that have shown that HACE1 is capable of tagging the Rho GTPase Rac1 for ubiquitin-mediated proteasomal degradation. In mammary epithelial cells, the loss of HACE1 leads to enhanced levels of active Rac1 resulting in increased clonogenicity, migration and invasion. Importantly, we show that targeting Rac1 can rescue the effects of HACE1 loss in mammary epithelial cells. Our results establish HACE1 as a breast cancer tumor suppressor gene by attenuating active Rac1 signaling. Our work supports the role of Rac1 as a critical signaling node in breast cancer and that loss of HACE1 leads to enhanced Rac1 signaling resulting in driving cancer progression. Furthermore, our results suggest the role of HACE1 loss as a biomarker for tumor progression and may identify patients vulnerable to Rac1 or Rac effector targeted therapies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-06.