ABSTRACT
Triple-negative breast cancer (TNBC) commonly develops resistance to chemotherapy, yet markers predictive of chemoresistance in this disease are lacking. Here, we define WNT10B-dependent biomarkers for ß-CATENIN/HMGA2/EZH2 signaling predictive of reduced relapse-free survival. Concordant expression of HMGA2 and EZH2 proteins is observed in MMTV-Wnt10bLacZ transgenic mice during metastasis, and Hmga2 haploinsufficiency decreased EZH2 protein expression, repressing lung metastasis. A novel autoregulatory loop interdependent on HMGA2 and EZH2 expression is essential for ß-CATENIN/TCF-4/LEF-1 transcription. Mechanistically, both HMGA2 and EZH2 displaced Groucho/TLE1 from TCF-4 and served as gatekeepers for K49 acetylation on ß-CATENIN, which is essential for transcription. In addition, we discovered that HMGA2-EZH2 interacts with the PRC2 complex. Absence of HMGA2 or EZH2 expression or chemical inhibition of Wnt signaling in a chemoresistant patient-derived xenograft (PDX) model of TNBC abolished visceral metastasis, repressing AXIN2, MYC, EZH2, and HMGA2 expression in vivo. Combinatorial therapy of a WNT inhibitor with doxorubicin synergistically activated apoptosis in vitro, resensitized PDX-derived cells to doxorubicin, and repressed lung metastasis in vivo. We propose that targeting the WNT10B biomarker network will provide improved outcomes for TNBC. SIGNIFICANCE: These findings reveal targeting the WNT signaling pathway as a potential therapeutic strategy in triple-negative breast cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/5/982/F1.large.jpg.
Subject(s)
Proto-Oncogene Proteins/metabolism , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/metabolism , Wnt Proteins/metabolism , Acetylation , Alleles , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Biomarkers, Tumor , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Doxorubicin/administration & dosage , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , Drug Synergism , Enhancer of Zeste Homolog 2 Protein/biosynthesis , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Female , HMGA2 Protein/biosynthesis , HMGA2 Protein/genetics , HMGA2 Protein/metabolism , Humans , Lymphoid Enhancer-Binding Factor 1 , Mice , Mice, Transgenic , Middle Aged , Neoplasm Metastasis , Pyrimidinones/administration & dosage , Pyrimidinones/pharmacology , Survival Rate , Transcription Factor 4 , Triple Negative Breast Neoplasms/genetics , beta Catenin/metabolismABSTRACT
A body of epidemiological evidence implicates exposure to endocrine disrupting chemicals (EDCs) with increased susceptibility to breast cancer. To evaluate the physiological effects of a suspected EDC in vivo, we exposed MCF-7 breast cancer cells and a patient-derived xenograft (PDX, estrogen receptor positive) to physiological levels of methylparaben (mePB), which is commonly used in personal care products as a preservative. mePB pellets (4.4 µg per day) led to increased tumor size of MCF-7 xenografts and ER+ PDX tumors. mePB has been thought to be a xenoestrogen; however, in vitro exposure of 10 nM mePB failed to increase MCF-7 cell proliferation or induction of canonical estrogen-responsive genes (pS2 and progesterone receptor), in contrast to 17ß-estradiol (E2) treatment. MCF-7 and PDX-derived mammospheres exhibited increased size and up-regulation of canonical stem cell markers ALDH1, NANOG, OCT4 and SOX2 when exposed to mePB; these effects were not observed for MDA-MB-231 (ER- ) mammospheres. As tumor-initiating cells (TICs) are also believed to be responsible for chemoresistance, mammospheres were treated with either tamoxifen or the pure anti-estrogen fulvestrant in the presence of mePB. Blocking the estrogenic response was not sufficient to block NANOG expression in mammospheres, pointing to a non-classic estrogen response or an ER-independent mechanism of mePB promotion of mammosphere activity. Overall, these results suggest that mePB increases breast cancer tumor proliferation through enhanced TIC activity, in part via regulation of NANOG, and that mePB may play a direct role in chemoresistance by modulating stem cell activity. Copyright © 2016 John Wiley & Sons, Ltd.
Subject(s)
Breast Neoplasms/chemically induced , Breast Neoplasms/genetics , Carcinogens/toxicity , Endocrine Disruptors/toxicity , Neoplastic Stem Cells/drug effects , Parabens/toxicity , Receptors, Estrogen/genetics , Animals , Carcinogens/antagonists & inhibitors , Cell Proliferation/drug effects , Estradiol/pharmacology , Estrogen Antagonists/pharmacology , Female , Humans , MCF-7 Cells , Mice , Mice, Nude , Neoplasm Proteins/genetics , Ovariectomy , Xenograft Model Antitumor AssaysABSTRACT
Osteoporosis is a disease characterized by low bone mass, leading to an increased risk of fragility fractures. GATA4 is a zinc-finger transcription factor that is important in several tissues, such as the heart and intestines, and has recently been shown to be a pioneer factor for estrogen receptor alpha (ERα) in osteoblast-like cells. Herein, we demonstrate that GATA4 is necessary for estrogen-mediated transcription and estrogen-independent mineralization in vitro. In vivo deletion of GATA4, driven by Cre-recombinase in osteoblasts, results in perinatal lethality, decreased trabecular bone properties, and abnormal bone development. Microarray analysis revealed GATA4 suppression of TGFß signaling, necessary for osteoblast progenitor maintenance, and concomitant activation of BMP signaling, necessary for mineralization. Indeed, pSMAD1/5/8 signaling, downstream of BMP signaling, is decreased in the trabecular region of conditional knockout femurs, and pSMAD2/3, downstream of TGFß signaling, is increased in the same region. Together, these experiments demonstrate the necessity of GATA4 in osteoblasts. Understanding the role of GATA4 to regulate the tissue specificity of estrogen-mediated osteoblast gene regulation and estrogen-independent bone differentiation may help to develop therapies for postmenopausal osteoporosis.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Estrogen Receptor alpha/metabolism , GATA4 Transcription Factor/metabolism , Osteoblasts/metabolism , Osteogenesis/physiology , Signal Transduction/physiology , Transforming Growth Factor beta/metabolism , Animals , Bone Morphogenetic Proteins/genetics , Cell Differentiation/physiology , Cells, Cultured , Estrogen Receptor alpha/genetics , GATA4 Transcription Factor/genetics , Gene Expression Regulation/physiology , Mice , Mice, Transgenic , Osteoblasts/cytology , Smad Proteins/genetics , Smad Proteins/metabolism , Transforming Growth Factor beta/geneticsABSTRACT
Wnt/ß-catenin signalling has been suggested to be active in basal-like breast cancer. However, in highly aggressive metastatic triple-negative breast cancers (TNBC) the role of ß-catenin and the underlying mechanism(s) for the aggressiveness of TNBC remain unknown. We illustrate that WNT10B induces transcriptionally active ß-catenin in human TNBC and predicts survival-outcome of patients with both TNBC and basal-like tumours. We provide evidence that transgenic murine Wnt10b-driven tumours are devoid of ERα, PR and HER2 expression and can model human TNBC. Importantly, HMGA2 is specifically expressed during early stages of embryonic mammogenesis and absent when WNT10B expression is lost, suggesting a developmentally conserved mode of action. Mechanistically, ChIP analysis uncovered that WNT10B activates canonical ß-catenin signalling leading to up-regulation of HMGA2. Treatment of mouse and human triple-negative tumour cells with two Wnt/ß-catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation. We demonstrate that WNT10B has epistatic activity on HMGA2, which is necessary and sufficient for proliferation of TNBC cells. Furthermore, HMGA2 expression predicts relapse-free-survival and metastasis in TNBC patients.
