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HER2 Signaling Drives DNA Anabolism and Proliferation Through SRC-3 Phosphorylation and E2F1-Regulated Genes Bryan C

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HER2 Signaling Drives DNA Anabolism and Proliferation Through SRC-3 Phosphorylation and E2F1-Regulated Genes Bryan C Published OnlineFirst February 1, 2016; DOI: 10.1158/0008-5472.CAN-15-2383 Cancer Molecular and Cellular Pathobiology Research HER2 Signaling Drives DNA Anabolism and Proliferation through SRC-3 Phosphorylation and E2F1-Regulated Genes Bryan C. Nikolai1, Rainer B. Lanz1, Brian York1, Subhamoy Dasgupta1, Nicholas Mitsiades1,2,3, Chad J. Creighton2,4, Anna Tsimelzon5, Susan G. Hilsenbeck5, David M. Lonard1, Carolyn L. Smith1, and Bert W. O'Malley1 Abstract Approximately 20% of early-stage breast cancers display scripts regulated by HER2 signaling are highly enriched with amplification or overexpression of the ErbB2/HER2 oncogene, E2F1 binding sites and define a gene signature associated with conferring poor prognosis and resistance to endocrine therapy. proliferative breast tumor subtypes, cell-cycle progression, and þ Targeting HER2 tumors with trastuzumab or the receptor DNA replication. We show that HER2 signaling promotes tyrosine kinase (RTK) inhibitor lapatinib significantly improves breast cancer cell proliferation through regulation of E2F1- survival, yet tumor resistance and progression of metastatic driven DNA metabolism and replication genes together with disease still develop over time. Although the mechanisms of phosphorylation and activity of the transcriptional coactivator cytosolic HER2 signaling are well studied, nuclear signaling SRC-3. Furthermore, our analyses identified a cyclin-dependent components and gene regulatory networks that bestow kinase (CDK) signaling node that, when targeted using the therapeutic resistance and limitless proliferative potential are CDK4/6 inhibitor palbociclib, defines overlap and divergence incompletely understood. Here, we use biochemical and bio- of adjuvant pharmacologic targeting. Importantly, lapatinib informatic approaches to identify effectors and targets of HER2 and palbociclib strictly block de novo synthesis of DNA, mostly transcriptional signaling in human breast cancer. Phosphory- through disruption of E2F1 and its target genes. These results lation and activity of the Steroid Receptor Coactivator-3 (SRC- have implications for rational discovery of pharmacologic 3) is reduced upon HER2 inhibition, and recruitment of SRC-3 combinations in preclinical models of adjuvant treatment and to regulatory elements of endogenous genes is impaired. Tran- therapeutic resistance. Cancer Res; 76(6); 1–13. Ó2016 AACR. Introduction domain proteins that function, in part, as signal adaptors and amplifiers for downstream kinase activity. Ras/Raf/MAPK The ErbB/HER receptor tyrosine kinase (RTK) family of mem- and PI3K/AKT pathways are subsequently activated and the brane growth factor receptors activates multiple signaling path- cascading substrates of these phospho-signaling events become ways and is often associated with cancer. Tyrosine kinase activity effectors of cell division, evasion of apoptosis, and general tumor- in this family of receptors is accomplished through homo- or igenicity (1, 2). heterodimerization of ErbB family members (EGFR/ERBB1, Steroid Receptor Coactivator-3 (SRC-3/AIB1/NCOA3) is a HER2/ERBB2, ERBB3, and ERBB4) upon ligand binding and/or potent transcriptional coregulator for various nuclear receptors membrane juxtaposition, and subsequent auto-and cross-phos- and transcription factors, including E2F1, NFkB, and members of phorylation of intracellular domains (1). Phosphorylated recep- the ETS family (3–6). SRC-3 is amplified and/or overexpressed in tor tyrosine residues serve as docking platforms for various SH2 a wide variety of tumors, including amplification in up to 10% and overexpression in up to 60% of breast tumors (3, 7–10). Importantly, SRC-3 is a coactivating partner for estrogen receptor 1 Department of Molecular and Cellular Biology, Baylor College of (ERa), the cognate effector for the mitogenic action of estrogenic Medicine, Houston, Texas. 2Department of Medicine, Baylor College of Medicine, Houston, Texas. 3Center for Drug Discovery, Baylor steroids in the mammary epithelium (11). ERa expression is an College of Medicine, Houston, Texas. 4Dan L. Duncan Cancer Center important biomarker for prognosis and treatment and is present Division of Biostatistics, Baylor College of Medicine, Houston, Texas. in 70% of breast tumors at diagnosis, most of which receive 5Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, Texas. endocrine therapy to block estrogen synthesis or receptor activity (12, 13). The function of SRC-3 in breast cancer has been inten- Note: Supplementary data for this article are available at Cancer Research a Online (http://cancerres.aacrjournals.org/). sively studied because it is a major coactivator for ER and a prominent, overexpressed oncogene. The coactivation potential C.L. Smith and B.W. O'Malley contributed equally to this article. of SRC-3 is tightly regulated by posttranslational modifications Corresponding Author: Bert W. O'Malley, Department of Molecular and Cellular that alter protein activity, stability, and subcellular localization Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. (14, 15). In the context of ERa and NFkB-regulated transcription, Phone: 713-798-6205; Fax: 713-798-5599; E-mail: [email protected] SRC-3 is differentially activated via phosphorylation at multiple doi: 10.1158/0008-5472.CAN-15-2383 residues in response to estradiol or TNFa (14, 16). Collectively, Ó2016 American Association for Cancer Research. these studies position SRC-3 as a potential "signal integrator" of www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst February 1, 2016; DOI: 10.1158/0008-5472.CAN-15-2383 Nikolai et al. various cytokine and hormone elicited cell-signaling events that Sample preparation, immunoblotting, and RT-PCR specify distinct patterns of gene expression (17). For protein analysis, cells were lysed (20 mmol/L Tris–HCl pH Clinical studies have shown that breast cancer patients with 8.0, 150 mmol/L NaCL, 1 mmol/L EDTA, and 0.5% NP-40) for 1 tumors expressing high levels of both HER2 and SRC-3 display hour at 4C, followed by centrifugation at 12,000 Â g for 10 resistance to endocrine therapy and have reduced disease-free minutes. The supernatant was removed to a fresh tube and diluted survival (12, 18, 19). Furthermore, SRC-3 knockout mice are for quantitation using the Bradford method. Samples were sep- resistant to HER2/neu-mediated breast tumorigenesis and dis- arated using NuPAGE 4% to 12% Bis–Tris gels (Life Technologies) play significantly reduced phosphorylation of key signaling and transferred to nitrocellulose using methanol transfer buffer. components, including HER2, AKT, and JNK (20). In MCF-7 Membranes were blocked in 5% fat-free milk reconstituted in PBS breast cancer cells, SRC-3 phosphorylation is influenced by plus 0.5% Tween-20 (PBST) for 1 hour at room temperature. HER2 (21). Together, these studies suggest HER2 and SRC-3 Primary antibodies were diluted in 1% fat-free milk in PBST are cooperating oncogenes whereby HER2 signaling events and blots incubated for several hours at room temperature or influence SRC-3 activity on discrete gene programs. However, overnightat4C. Species-specific secondary antibodies conju- the transcriptional targets of HER2 and SRC-3 are largely gated with horseradish peroxidase were used with traditional undescribed and it is unknown whether intrinsic SRC-3 activity enhanced chemiluminescense and X-ray film. Densitometric and specific phosphorylation sites are affected by HER2. Ampli- quantification was performed using ImageJ and numbers in fication of HER2 occurs in approximately 20% of all breast figures represent the area under curve for each band, normal- tumors, and overexpression of HER2 and SRC-3 is associated ized to actin in the identical lane. TRIzol reagent (low pH with endocrine resistance and extremely poor prognosis. Given phenol; Life Technologies) was used to collect and extract total the link between HER2 signaling and SRC-3 in breast cancer RNA. RNA concentration was determined using a NanoDrop and the importance of SRC-3 phosphorylation for its coactiva- spectrometer and integrity was evaluated using Lab-on-chip tion of steroid receptors and other transcription factors, the (Agilent). Reverse transcription using random hexamer priming combined effects of HER2 and SRC-3 on gene regulation are (BioRad iScript) was performed before qPCR on ABI OneStep understudied in breast cancer. Additionally, nuclear effectors of with Roche Universal Library hydrolysis probes. Data were HER2 signaling that regulate transcription of tumorigenic gene analyzed using the DDCT method (see www.nursa.org/qpcr_tu- networks are incompletely understood. torials/ for review). Detailed descriptions of primer sequences Here, we study the effects of HER2-mediated signaling events are listed in Supplementary Table S2. on SRC-3 activity in breast cancer cells with amplified HER2 and SRC-3 genes. HER2 depletion attenuates site-specificphos- RNAi, transfections, and luciferase assays phorylation of SRC-3, leading to a reduction in its intrinsic siRNA was custom designed and/or ordered as prevalidated activity. Transcriptomic analysis identifies genes that are regu- duplexes (Life Technologies; see Supplementary Table S2 for lated by HER2 signaling, with a massive enrichment for tran- details). BT-474 cells were plated on 6-well plates at a density of scripts involved in mitotic cell-cycle processes and DNA repli- 4 Â 105 per well 1 day prior to transfection
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