Oncogene (2015) 34, 1160–1173 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE PKA signaling drives mammary tumorigenesis through Src AG Beristain1, SD Molyneux1,2, PA Joshi1,2, NC Pomroy1, MA Di Grappa1, MC Chang3, LS Kirschner4, GG Privé2, MA Pujana5 and R Khokha1,2,3 Protein kinase A (PKA) hyperactivation causes hereditary endocrine neoplasias; however, its role in sporadic epithelial cancers is unknown. Here, we show that heightened PKA activity in the mammary epithelium generates tumors. Mammary-restricted biallelic ablation of Prkar1a, which encodes for the critical type-I PKA regulatory subunit, induced spontaneous breast tumors characterized by enhanced type-II PKA activity. Downstream of this, Src phosphorylation occurs at residues serine-17 and tyrosine-416 and mammary cell transformation is driven through a mechanism involving Src signaling. The phenotypic consequences of these alterations consisted of increased cell proliferation and, accordingly, expansion of both luminal and basal epithelial cell populations. In human breast cancer, low PRKAR1A/high SRC expression defines basal-like and HER2 breast tumors associated with poor clinical outcome. Together, the results of this study define a novel molecular mechanism altered in breast carcinogenesis and highlight the potential strategy of inhibiting SRC signaling in treating this cancer subtype in humans. Oncogene (2015) 34, 1160–1173; doi:10.1038/onc.2014.41; published online 24 March 2014 INTRODUCTION of Prkar1a in mesenchymal lineage cells is sufficient for 18 Cyclic-AMP dependent protein kinase A (PKA) ubiquitously spontaneous osteosarcoma development. However, the role of functions as a signaling hub downstream of G-protein coupled PKA signaling in other cancer types and, particularly, in mammary receptors and cAMP to regulate a spectrum of biological processes carcinogenesis remains unknown. across tissues.1–6 PKA impacts multiple signaling networks in both In this study, we show that altered PKA regulation leading to physiological and pathological conditions by phosphorylating increased PKA activity in mammary tissue promotes carcino- target proteins on serine/threonine residues. The complexity genesis. Prkar1a loss results in heightened PKA activity defined by associated with PKA function stems from its presence as two an increase in type-II PKA isozyme in mammary epithelial cells and distinct heterotetramers, termed type-I and type-II PKA,4,7 with this hyperactivation drives mammary cell transformation through each PKA isozyme varying with respect to protein subunit a mechanism involving Src. We further find that low PRKAR1A/high composition, cellular localization and turnover. Four regulatory SRC marks a tumor subset of poor-prognosis basal-like and HER2 (R) subunits (R1α,R1β,R2α and R2β) and four catalytic (C) subunits breast cancer. (Cα,Cβ,Cγ and Prkx) have been identified, where the presence of R1 or R2 subunits defines the type of PKA isozyme as type-I or RESULTS type-II, respectively.8 The balance between type-I/-II PKA can influence cell cycle entry and terminal differentiation in multiple Prkar1a loss in the mammary gland is sufficient to cause systems.4,7 Dysregulated PKA activity leads to the development of mammary tumors tumors in cAMP-responsive endocrine tissues9,10 and this is To explore the effects of PKA hyperactivation in epithelial cells, thought to stem from imbalances in activities of either type-I or we selected the mammary gland, a tissue outside of classical type-II PKA;7,11 however, its role as a cancer driver in a wider endocrine epithelium harboring an extensive ductal network with spectrum of tissues is less well known. marker-defined lineages. It can develop a diverse family of The discovery of autosomal dominant inactivating mutations of molecular cancer subtypes in humans.19,20 Several mouse models the PRKAR1A gene as the cause of Carney complex syndrome first are available, including those that allow homozygous gene linked PKA dysregulation to carcinogenesis.12,13 PRKAR1A encodes deletion in the majority of mammary ductal epithelium. We the PKA regulatory subunit R1α, and of the four PKA regulatory adopted a genetic strategy (Figure 1a) in which mammary-specific subunits, only PRKAR1A is essential for tissue development and deletion of Prkar1a was created by crossing Prkar1alox/lox mice cAMP-dependent regulation.14 Mutations to this gene in humans (Prkar1a exon 2 flanked by LoxP sites) with transgenic mice and mice induce multiple endocrine tumors as well as myxomas, expressing Cre recombinase under the mammary epithelial- osteoblastic neoplasias and schwannomas.9,15 When combined specific MMTV promoter. Unexpectedly, MMTV-Cre deletion of À À À with Tp53+/ or Rb1+/ backgrounds, Prkar1a+/ mice exhibit a Prkar1a was sufficient to generate mammary tumors (Figure 1b); generally increased incidence of sarcomas, pituitary tumors, Cre-mediated excision of the Prkar1a gene in this cohort of mice is thyroid tumors and chemically induced skin papillomas.10,16,17 In shown in Figure 1c. Prkar1aΔMam mice developed multiple tumors addition, we have found that tissue-specific heterozygous deletion with 100% penetrance and a latency of 9–15 months of age (18/18 1Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Ontario, Canada; 2Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; 3Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 4Division of Endocrinology, Diabetes and Metabolism, The Ohio State University, Columbus, OH, USA and 5Breast Cancer and Systems Biology Unit, Translational Research Laboratory, Catalan Institute of Oncology, IDIBELL, L’Hospitalet del Llobregat, Barcelona, Spain. Correspondence: Dr R Khokha, Department of Medical Biophysics and Department of Laboratory Medicine and Pathobiology, Ontario Cancer Institute, University of Toronto, Toronto, Ontario, Canada M5G 2M9. E-mail: [email protected] Received 1 August 2013; revised 20 December 2013; accepted 24 December 2013; published online 24 March 2014 PKA-induced Src drives mammary tumorigenesis AG Beristain et al 1161 Figure 1. Conditional loss of Prkar1a is sufficient to generate mammary tumors. (a) Schematic describes mouse-breeding strategy for the generation of Prkar1aΔMam mice. Prkar1aΔMam (MMTV-Cre/Prkar1afl/fl) denotes homozygous deletion by Cre recombinase expressed under the control of the MMTV promoter. (b) Survival plot of Prkar1aΔMam (n = 18; solid black line) and control MMTV-Cre mice (n = 6; dashed line). (c) Cre-mediated genomic excision of Prkar1a in primary mammary epithelial cells derived from Prkar1aΔMam mice assessed by PCR; MMTV-Cre mammary epithelial cells do not exhibit Cre-directed Prkar1a excision shown by lack of 175-bp PCR product. Prkar1afl/+ mouse osteoblast cultures transduced with retroviral Cre-recombinase (p-Cre) or GFP (p-GFP) serve as positive or negative controls.19 ‘L’ indicates DNA ladder. (d) Mammary gland whole-mounts of Prkar1aΔMam from 1.4 to 13 months of age highlights tissue progression to tumors. LN denotes lymph node; red arrows highlight progression to tumors, ‘mo’ indicates age in months. Representative (e) H&E staining of tumors from Prkar1aΔMam mice showing papillary, mixed (mix) and invasive ductal carcinoma (IDC) mammary tumors. Scale bars, 100 μm. (f) Representative immunofluorescent images of Prkar1aΔMam mammary tumors dual-labeled with epithelial lineage markers keratin 14 (basal) and keratin 18 (luminal). Merged images show the combination of keratin 14 (red), keratin 18 (green) and DAPI (blue) positivity. Scale bars, 50 μm. See also Supplementary Figure 1 mice aged o16 months; Figure 1b; Supplementary Figure 1A) mammary tumors showed immunohistochemical positivity for and progression from ductal hyperplasia (4.2 months) to palpable estrogen receptor α (Erα) and progesterone receptor (Pgr), tumors (13 months) was observed by whole-mount analysis whereas less-differentiated tumors harboring an invasive pheno- (Figure 1d). type were immuno-negative for Erα (Supplementary Figure 1B). Histologically, mammary glands of >10 month-old Prkar1aΔMam Next, to examine whether Prkar1a loss cooperates with mice had an abundance of lobular hyperplasia, back-to-back molecular pathways known to be activated in human breast growth and areas of atypia (Figure 1e). The tumors had cancer, Prkar1aΔMam mice were bred into the widely used MMTV- characteristics of papillomas with gradual progression to ductal PyMT (polyoma virus middle T-antigen) model that induces carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) activation of ErbB2, Src, c-Myc and Ras/PI3 kinase signaling (Figure 1e). In the majority of mammary tumors, a profound networks (Figure 2a).21,22 Confirmation of Cre-mediated Prkar1a expansion of keratin 18-positive luminal cells was observed, which gene excision in this cohort was confirmed by PCR (Figure 2b). was accompanied with scattered expression of the myoepithelial Tumor development was faster, with increased tumor burden, in marker keratin 14 (Figure 1f). Additionally, select Prkar1aΔMam Prkar1aΔMam/PyMT mice compared with PyMT controls (Figures 2). © 2015 Macmillan Publishers Limited Oncogene (2015) 1160 – 1173 PKA-induced Src drives mammary tumorigenesis AG Beristain et al 1162 Figure 2. Prkar1a ablation in the PyMT mammary tumor model accelerates tumorigenesis. (a) Schematic describes mouse-breeding strategy for the generation of Prkar1aΔMam/PyMT mice. (b) Cre-mediated