Oncogene (2014), 1–14 © 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 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 advance online publication, 24 March 2014; doi:10.1038/onc.2014.41

INTRODUCTION Cyclic-AMP dependent protein kinase A (PKA) ubiquitously functions as a signaling hub downstream of G-protein coupled receptors and cAMP to regulate a spectrum of biological processes across tissues.1–6 PKA impacts multiple signaling networks in both physiological and pathological conditions by phosphorylating target proteins on serine/threonine residues. The complexity associated with PKA function stems from its presence as two distinct heterotetramers, termed type-I and type-II PKA,4,7 with each PKA isozyme varying with respect to protein subunit composition, cellular localization and turnover. Four regulatory (R) subunits (R1α, R1β, R2α and R2β) and four catalytic (C) subunits (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 type-II, respectively.8 The balance between type-I/-II PKA can influence cell cycle entry and terminal differentiation in multiple systems.4,7 Dysregulated PKA activity leads to the development of tumors in cAMP-responsive endocrine tissues9,10 and this is thought to stem from imbalances in activities of either type-I or type-II PKA;7,11 however, its role as a cancer driver in a wider spectrum of tissues is less well known. The discovery of autosomal dominant inactivating mutations of the PRKAR1A gene as the cause of Carney complex syndrome first linked PKA dysregulation to carcinogenesis.12,13 PRKAR1A encodes the PKA regulatory subunit R1α, and of the four PKA regulatory subunits, only PRKAR1A is essential for tissue development and cAMP-dependent regulation.14 Mutations to this gene in humans and mice induce multiple endocrine tumors as well as myxomas, osteoblastic neoplasias and schwannomas.9,15 When combined with Tp53+/
or Rb1+/
backgrounds, Prkar1a+/
mice exhibit a generally increased incidence of sarcomas, pituitary tumors, thyroid tumors and chemically induced skin papillomas.10,16,17 In addition, we have found that tissue-specific heterozygous deletion

of Prkar1a in mesenchymal lineage cells is sufficient for spontaneous osteosarcoma development.18 However, the role of PKA signaling in other cancer types and, particularly, in mammary carcinogenesis remains unknown. In this study, we show that altered PKA regulation leading to increased PKA activity in mammary tissue promotes carcinogenesis. Prkar1a loss results in heightened PKA activity defined by an increase in type-II PKA isozyme in mammary epithelial cells and this hyperactivation drives mammary cell transformation through a mechanism involving Src. We further find that low PRKAR1A/high SRC marks a tumor subset of poor-prognosis basal-like and HER2 breast cancer. RESULTS Prkar1a loss in the mammary gland is sufficient to cause mammary tumors To explore the effects of PKA hyperactivation in epithelial cells, we selected the mammary gland, a tissue outside of classical endocrine epithelium harboring an extensive ductal network with marker-defined lineages. It can develop a diverse family of molecular cancer subtypes in humans.19,20 Several mouse models are available, including those that allow homozygous gene deletion in the majority of mammary ductal epithelium. We adopted a genetic strategy (Figure 1a) in which mammary-specific deletion of Prkar1a was created by crossing Prkar1alox/lox mice (Prkar1a exon 2 flanked by LoxP sites) with transgenic mice expressing Cre recombinase under the mammary epithelialspecific MMTV promoter. Unexpectedly, MMTV-Cre deletion of Prkar1a was sufficient to generate mammary tumors (Figure 1b); Cre-mediated excision of the Prkar1a gene in this cohort of mice is shown in Figure 1c. Prkar1aΔMam mice developed multiple tumors with 100% penetrance and a latency of 9–15 months of age (18/18

1 Ontario 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

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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 o 16 months; Figure 1b; Supplementary Figure 1A) and progression from ductal hyperplasia (4.2 months) to palpable tumors (13 months) was observed by whole-mount analysis (Figure 1d). Histologically, mammary glands of >10 month-old Prkar1aΔMam mice had an abundance of lobular hyperplasia, back-to-back growth and areas of atypia (Figure 1e). The tumors had characteristics of papillomas with gradual progression to ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) (Figure 1e). In the majority of mammary tumors, a profound expansion of keratin 18-positive luminal cells was observed, which was accompanied with scattered expression of the myoepithelial marker keratin 14 (Figure 1f). Additionally, select Prkar1aΔMam Oncogene (2014), 1 – 14

mammary tumors showed immunohistochemical positivity for estrogen receptor α (Erα) and progesterone receptor (Pgr), whereas less-differentiated tumors harboring an invasive phenotype were immuno-negative for Erα (Supplementary Figure 1B). Next, to examine whether Prkar1a loss cooperates with molecular pathways known to be activated in human breast cancer, Prkar1aΔMam mice were bred into the widely used MMTVPyMT (polyoma virus middle T-antigen) model that induces activation of ErbB2, Src, c-Myc and Ras/PI3 kinase signaling networks (Figure 2a).21,22 Confirmation of Cre-mediated Prkar1a gene excision in this cohort was confirmed by PCR (Figure 2b). Tumor development was faster, with increased tumor burden, in Prkar1aΔMam/PyMT mice compared with PyMT controls (Figures 2). © 2014 Macmillan Publishers Limited

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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 genomic excision of Prkar1a in primary mammary epithelial cells derived from Prkar1aΔMam/PyMT mice assessed by PCR; MMTV-PyMT mammary epithelial cells (PyMT) do not display Cre-directed Prkar1a excision shown by lack of 175-bp PCR product. Prkar1afl/+ mouse osteoblast cultures transduced with Cre-recombinase (p-Cre) or GFP (p-GFP) serve as positive or negative controls.19 ‘L’ indicates DNA ladder. (c) Combined inguinal mammary gland weights (mg) of Prkar1aΔMam/PyMT (n = 8) and PyMT (n = 12) at 40 and 70 days of age. *P o0.05. (d) Survival plot for Prkar1aΔMam/PyMT (n = 15; red line) and PyMT (n = 14; gray line) mice. Representative gross (e), whole mount (f) and H&E (g) images of 40-day-old inguinal mammary glands of Prkar1aΔMam/PyMT and PyMT mice. Dotted line and red arrows highlight tumor periphery and location. (h) Ki67 immunohistochemistry in mammary tissue from age-matched 40-day-old Prkar1aΔMam/PyMT and PyMT, as well as in 55-day-old PyMT mammary tumor. Bars, 100 μm. See also Supplementary Figure 2.

In these mice, tumors were palpable by 33 days of age, reaching end point earlier compared with PyMT controls (detection median, 38 versus 70 days; end point median, 72 versus 95 days), as shown in Figure 2d and Supplementary Figure 2. Gross tumor images and mammary gland whole mounts displayed extensive hyperplasia as early as 40 days of age in Prkar1aΔMam/PyMT mice (Figures 2). This demonstrates that mammary tumorigenesis is accelerated upon conditional deletion of Prkar1a in an established breast cancer model. Tumors in Prkar1aΔMam/PyMT cohort were classified as DCIS harboring similar characteristics to those known to develop in the PyMT model (Figure 2g). Forty-day-old lesions in Prkar1aΔMam/PyMT had far greater Ki67 positivity than in control cohorts at 40 or 55 day of age, indicating that mammary deletion of Prkar1a drives cellular proliferation (Figure 2h). Taken together, we demonstrate that mammary-specific deletion of Prkar1a is sufficient to induce stochastic mammary tumorigenesis exhibiting step-wise stages of histological progression. Further, in the aggressive MMTV-PyMT mouse model of breast cancer, Prkar1a loss profoundly accelerates mammary tumorigenesis. Our data show that Prkar1a have a tumor suppressor-like function in the mammary epithelium. Mammary epithelial lineage expansion upon PKA hyperactivation We and others have demonstrated that loss of the Prkar1a PKA regulatory subunit results in elevated PKA enzymatic activity in bone and endocrine tissues.18,23 We sought to test whether this occurs in mammary epithelium, following Prkar1a tissue-specific ablation. Analyzing basal (unstimulated) and total (cAMP-induced) © 2014 Macmillan Publishers Limited

PKA activity in protein lysates from age-matched non-tumor (MMTV-Cre) and tumor-bearing (Prkar1aΔMam) mammary tissue revealed significantly higher baseline PKA activity in Prkar1aΔMam relative to MMTV-Cre controls (P = 0.047). Treatment with the PKA inhibitor, PKI, abolished the effect of cAMP-induced PKA activity demonstrating kinase specificity of the assay (Figure 3a). In addition, baseline and total PKA activity increased four- to fivefold in 40-day Prkar1aΔMam/PyMT tumors compared with age-matched PyMT mammary tissue lysates (Figure 3b). Notably, PKA activity in non-tumor bearing 40-day-old mammary glands of Prkar1aΔMam mice or 55-day-old PyMT mammary tumors was not elevated. Accordingly, PyMT/Prkar1aΔMam tumors at 40 and 90 days of age exhibited elevated phosphorylated CREB, a canonical downstream target of PKA (Figures 3). We next explored the cellular makeup of Prkar1aΔMam mammary tumors by flow cytometry to determine the effect of PKA hyperactivation on distinct mammary epithelial lineages. This analysis revealed a significant expansion of both the luminal CD24 +/CD49flo and basal CD24+/CD49fhi epithelial subpopulations (Figure 3e). The basal fraction, known to contain mammary stem cells,24 was increased to >13% of total cells compared with 5% in control MMTV-Cre tissue. Luminal-type breast cancers that develop in the PyMT model are reported to exhibit a profound expansion of luminal (CD24+/CD49flo) mammary epithelial cells,21 and we observed a more extreme expansion of this population in the Prkar1aΔMam/PyMT compared with the PyMT cohort (Figure 3f). Biochemically, fluorescence-activated cell sorting (FACS)-purified CD24+/CD49flo luminal mammary epithelial cells Oncogene (2014), 1 – 14

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Figure 3. Mammary-specific deletion of Prkar1a results in elevated PKA activity and mammary epithelial cell expansion. (a) Quantification of total (cAMP-treated) and baseline (unstimulated) PKA activity in adult age-matched (10–13 months of age) littermate Prkar1aΔMam mammary tumors (n = 5) and control MMTV-Cre mammary glands (n = 4). PKI inhibitor co-treatment demonstrates PKA activity specificity. (b) Baseline and total PKA activity of age-matched 40-day-old Prkar1aΔMam/PyMT tumors (n = 4), tumor-free mammary tissues from Prkar1aΔMam (n = 1), MMTV-Cre (Wt; n = 1) and PyMT (n = 4) mice and 55-day-old (55 d) PyMT mammary tumors (n = 3)* P o0.05. (c) Phosphorylated Creb (Ser 133) and total Creb protein levels in mammary protein lysates from 40- and 90-day-old Prkar1aΔMam/PyMT and MMTV-Cre mice. Wt = age-matched MMTV-Cre control. Numbers indicate individual mice from each cohort. (d) Phosphorylated Creb immunohistochemistry in Prkar1aΔMam/PyMT mammary tumor and PyMT 40-day-old mammary gland. Bar, 100 μm. (e) Representative FACS plots segregated by cell surface markers CD24 and CD49f identifying luminal (L), basal (B) and stromal (S) subpopulations in adult (4–6 months) age-matched Prkar1aΔMam and control MMTV-Cre glands; percentage of cells representing each cell population is included. Bar graphs compare total numbers of luminal (lin
/CD24+/CD49flo) and basal (lin
/CD24+/CD49fhi) epithelial cells in each cohort. N = 5/cohort; *Po0.05. (f) FACS plots segregated by cell surface markers CD24 and CD49f identifying luminal (L), basal (B) and stromal (S) subpopulations in 40-day-old Prkar1aΔMam/PyMT and PyMT mammary tissues; percentage of cells representing each cell population is included. Bar graphs compare total numbers of luminal (lin
/CD24+/CD49flo) and basal (lin
/CD24+/CD49fhi) epithelial cells in each cohort. N = 5/cohort. *Po 0.05. (g) Baseline and total PKA activities in FACS-purified luminal mammary cells from PyMT (n = 3) and Prkar1aΔMam/PyMT (n = 3) mice. *P ⩽ 0.05.

from Prkar1aΔMam/PyMT showed increased baseline and total PKA activity (Figure 3g). These data demonstrate that mammaryspecific loss of Prkar1a elevates PKA activity and this coincides with substantial expansion of the luminal and basal epithelial subpopulations. Type-II PKA isozyme hyperactivation in Prkar1aΔMam tumors We next set out to determine the predominant PKA isozyme subtype in mammary tissues and mammary epithelial cells of Oncogene (2014), 1 – 14

Prkar1aΔMam and Prkar1aΔMam/PyMT mice. Gene expression analysis of PKA regulatory (Prkar1a, Prkar1b, Prkar2a and Prkar2b) and catalytic (Prkaca) subunits was performed using FACS-purified luminal, basal and stromal mammary cells. The lineage markers keratin 14 and keratin 18 were used to verify that sorted cells were purely basal (keratin 14-positive) or luminal (keratin 18-positive) (Figure 4a). As expected, Prkar1a expression was reduced in both the luminal and basal epithelial subpopulations from Prkar1aΔMam mice, while remaining unchanged in the stromal cell subpopulation, confirming the specificity of the MMTV-directed conditional © 2014 Macmillan Publishers Limited

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Figure 4. PKA-II isozyme defines Prkar1a mammary-specific loss. (a) qPCR expression analysis of keratin 14, keratin 18, Prkar1a, Prkar1b, Prkar2a, Prkar2b and Prkaca in luminal (L), basal (B) and stromal (S) FACS-purified cell subpopulations derived from adult age-matched Prkar1aΔMam mammary tumors and control MMTV-Cre mammary glands (n = 5 mice per cohort); *P o0.05 among individual cell populations. (b) Expression of Prkar1a, Prkar1b, Prkar2a, Prkar2b and Prkaca PKA subunits in flow-purified luminal mammary epithelial cells derived from 40-day-old age-matched Prkar1aΔMam/PyMT and PyMT mice (n = 5 mice/cohort); *Po0.05. β-actin was used for normalization in all cohorts. (c, d) Measurement of PKA activity in individual FPLC protein fractions and subsequent verification of PKA isozyme by western blotting. PKA activity in Prkar1aΔMam tumors (n = 3) and control MMTV-Cre mammary glands (n = 2) are shown in (c), while PKA activity in age-matched 40-day-old Prkar1aΔMam/PyMT and PyMT mammary tumors (n = 4/cohort) are in shown in (d). Specifically, protein lysates from tissues were fractionated by DEAE column chromatography over a linear salt gradient. Activity peaks correspond to type-I PKA and type-II PKA isozymes, which elute at different salt concentrations. Fraction numbers (14–44) are specified on the x axis, while kinase activities measured by radioactive counts/min are shown on the y axis. Western blots verify the holoenzyme identity of eluted PKA heterotetramers based on the antibody specific for R1α (type-I PKA) or R2β (type-II PKA) PKA regulatory subunits in FPLC-fractionated protein lysates. Non-fractionated protein lysates served as positive (input) controls.

gene deletion in the epithelial compartment (Figure 4a). Next, while Prkar1b expression did not change in any cell type, the expression of both Prkar2a and Prkar2b regulatory subunit genes increased significantly in luminal and basal subpopulations, but were unchanged in the stroma (Figure 4a). Expression of the catalytic subunit, Prkaca, was higher in all cellular subpopulations (Figure 4a). Given the luminal cell expansion observed in the PyMT model (Figure 3f), the expression of PKA subunits in this subpopulation was further analyzed in Prkar1aΔMam/PyMT mice. Consistent with the above observations, significant overexpression of both regulatory type-II and catalytic subunits was observed, while Prkar1a levels were reduced and Prkar1b levels unchanged in the luminal fraction (Figure 4b). Altogether, these changes suggest a shift towards the type-II PKA holoenzyme in mammary epithelium following the loss of Prkar1a. © 2014 Macmillan Publishers Limited

To biochemically profile PKA holoenzyme kinase activity, we fractionated total mammary tissue protein, and used a combination of kinase assays and immunoblotting to match PKA activity with type-I and type-II regulatory subunit abundance (Figures 4). Control MMTV-Cre and Prkar1aΔMam (Figure 4c) or PyMT and Prkar1aΔMam/PyMT (Figure 4d) protein lysates prepared from whole mammary tissues were subjected to anion exchange chromatography and activity assays were performed on each collected fraction. Immunoblotting against R1α and R2β proteins in fractionated samples confirmed the identity of each PKA holoenzyme species (type-I or type-II PKA) eluting across the linear salt gradient.15 In total profiles, MMTV-Cre mammary glands exhibited comparable amounts of type-I and -II PKA activity, with PyMT control glands showing more PKA type-I activity (Figures 4). Notably, Prkar1aΔMam and Prkar1aΔMam/PyMT Oncogene (2014), 1 – 14

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Figure 5. Aberrant PKA activity drives mammary cell proliferation. (a) Cell proliferation over 96 h time course of Prkar1aΔMam/PyMT mammary epithelial cells stimulated with 1.0 mM cAMP. (b) Images of 3D mammary epithelial cell colonies at day 8 of culture. Prkar1aΔMam/PyMT and PyMT cells were cultured in the presence of cAMP (1.0 mM), H89 (10 μM) or vehicle 1% DMSO (
). (c) Comparison of colonies formed between Prkar1aΔMam/PyMT and PyMT. Colonies were quantified based on the indicated area and averages from five fields of view were calculated. (d) PKA-R2β immunoprecipitates (IP) from Prkar1aΔMam/PyMT cells treated with control peptide (Control) or AKAP-StHT31 inhibitor (Ht31) immunoblotted (IB) for AKAP1; protein lysates from identical treatments were immunoblotted for AKAP1 or PKA-R2β. Molecular weights (kDa) are shown on the left. (e) Cell proliferation over 96 h time course of Prkar1aΔMam/PyMT mammary epithelial cells treated with 5 μM AKAP-StHt31 (Ht31), control peptide (Control) or left untreated (
). (f) Images of Prkar1aΔMam/PyMT 3D mammary epithelial cell colonies at day 8 of culture; cAMP-stimulated (cAMP) or unstimulated (
) cells were cultured with control peptide (Control) or AKAP-St-Ht31 inhibitor (Ht31). (g) Quantification of Prkar1aΔMam/PyMT colonies ⩾25mm2 in diameter. Colony diameters were measured and averaged from five fields of view. a = P ⩽ 0.05 when compared with cAMP-treated PyMT sample; b = P ⩽ 0.05 when compared with non-treated PyMT sample. * = P ⩽ 0.05 compared with control peptide. Cell proliferation and colony formation assays were performed in triplicate, and repeated three times. See also Supplementary Figure 3.

tumors exhibited dramatic increases in type-II PKA activity, and experienced a decrease in type-I PKA (Figures 4), as previously suggested by our mRNA expression analyses; modest type-I PKA activity observed in Prkar1aΔMam is likely from non-epithelial (stromal) contributions from homogenized mammary tissues. This series of gene/protein expression and kinase activity studies demonstrate that mammary Oncogene (2014), 1 – 14

epithelial deletion of Prkar1a results in type-II PKA isozyme hyperactivation. Heightened PKA activity drives mammary epithelial cell proliferation Having showed that Prkar1a ablation in mammary epithelium is sufficient to induce tumorigenesis, we next asked whether the © 2014 Macmillan Publishers Limited

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Figure 6. Mammary gland-specific loss of Prkar1a induces Src activation. (a) Phosphorylated PKA substrates from 40-day-old age-matched Prkar1aΔMam/PyMT or PyMT mammary tissues were immunoprecipitated (IP) using an antibody-specific for PKA-phosphorylated serine/ threonine (RRXS*/T*) epitopes, followed by immunoblotting (IB) with anti-Src antibody. Molecular weights (kDa) are shown on the left and numbers represent individual mice. (b) Immunoblots of phosphorylated and total protein levels of Src (Ser17; Tyr416) and Akt (Ser473) in mammary protein lysates. β-actin indicates loading control. Numbers represent individual mice. (c) Immunoprecipitation (IP) of phosphorylated PKA substrates from Prkar1aΔMam tumors and control MMTV-Cre mammary glands followed by Src immunoblotting (IB). Numbers indicate individual mice from respective cohorts. (d) Src Tyr416 phosphorylation indicates active Src in protein lysates from Prkar1aΔMam and control mammary tissue. Protein lysate from Prkar1aΔMam/PyMT tumor are a positive control. See also Supplementary Figure 4.

increases in PKA activity downstream of Prkar1a deletion are required for mammary epithelial proliferation. Primary mammary epithelial cells derived from Prkar1aΔMam/PyMT and control PyMT mice were subjected to proliferation assays stimulated with cAMP and treated with the PKA inhibitors H89 or PKI. To begin with, untreated Prkar1aΔMam/PyMT cells had higher baseline proliferation rates than PyMT cells over 96 h of culture (Figure 5a; Supplementary Figure 3A). The addition of cAMP enhanced proliferation in both groups; however, Prkar1aΔMam/PyMT cells were more sensitive to this stimulation, showing a two-fold induction in proliferation over wild-type PyMT cells (Figure 5a). The PKA inhibitors H89 and PKI abolished the effect of cAMP on mammary epithelial cell proliferation across the time course in both genotypes (Supplementary Figure 3). To assess the influence of PKA activity on cellular transformation, Prkar1aΔMam/PyMT and PyMT mammary epithelial cells were grown in Matrigel to assess the potential of colony formation. Cells from both models grew significantly more large colonies in the presence of cAMP (Figures 5). Importantly, Prkar1aΔMam/PyMT cells generated larger colonies than PyMT alone, even in the absence of cAMP, and this was abrogated upon H89 co-treatment. To directly examine the importance of elevated type-II PKA in driving cell proliferation and transformation, we treated Prkar1aΔMam/PyMT cells with the peptide inhibitor, AKAP St-Ht31, which prevents type-II PKA regulatory subunit interaction with A-kinase anchoring proteins (AKAPs)25,26 important for optimal activity and cellular localization.27,28 AKAP St-Ht31 peptide mediated disruption of PKA–R2AKAP interaction was verified by PKA–R2α immunoprecipitation followed by AKAP1 immunoblotting, which showed reduced R2βAKAP1 interaction (Figure 5d). Importantly, AKAP St-Ht31 peptide inhibitor treatment effectively decreased baseline proliferation and 3D colony formation, while treatment with a © 2014 Macmillan Publishers Limited

control peptide (St-Ht31P) had no effect (Figures 5). AKAP St-Ht31 modestly, but significantly inhibited cAMP-induced colony formation (Figures 5). Together, these experiments demonstrate that type-II PKA isozyme is fundamental in driving Prkar1aΔMam/PyMT cell growth. Thus, heightened type-II PKA activation is sufficient and required to promote mammary epithelial cell hyperproliferation in the context of Prkar1a deletion. PKA activates Src signaling during mammary tumorigenesis Given our findings that aberrant PKA activity regulates cell proliferation, we sought the molecular mechanism through which PKA drives breast carcinogenesis. To this end, we first investigated the Wnt and Erα signaling pathways, which are modulated downstream of PKA in other cancer types.5,10,29 Expression of several Wnt and Erα target genes (Axin2, Tcf4, Greb1 and Pra/b) were measured in 40-day-old Prkar1aΔMam/PyMT and control PyMT FACS-purified luminal cells. This analysis failed to reveal differences in the expression of these target genes between these cohorts (Supplementary Figure 4), suggesting that type-II PKA hyperactivity accelerates PyMT breast cancers by mechanisms other than Erα or Wnt pathways. PKA can phosphorylate c-Src (herein referred to as Src) on serine-17 to regulate its activity.30 Given that Src is involved in PyMT tumorigenesis,22,31 its activation was evaluated in 40-dayold PyMT cohort mammary tissues. PKA protein substrates were immunoprecipitated using an antibody specifically targeting phosphorylated serine/threonine epitopes (RRXS*/T*) of PKA substrates,32 followed by Src immunoblotting. This showed highly elevated PKA-phosphorylated Src across Prkar1aΔMam/PyMT mammary tissues (Figure 6a). Since Ser17 phosphorylation of Src by PKA directs its activation through auto-phosphorylation of Oncogene (2014), 1 – 14

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Tyr416,33 we examined whether this occurs in Prkar1aΔMam/PyMT mammary glands. Prkar1aΔMam/PyMT tissues exhibited consistently elevated Ser17 and Tyr416 Src phosphorylation (Figure 6b). Additionally, levels of total Src were also higher in Prkar1aΔMam/ PyMT mice. Further, phospho-Akt (Ser473), a signaling effector known to be downstream of Src, was probed and found to be higher in Prkar1aΔMam/PyMT (Figure 6b). To determine whether Src is also phosphorylated by PKA in Prkar1aΔMam tumors that develop spontaneously without the PyMT oncogene, we analyzed tissue bearing advanced tumors from these mice. Immunoprecipitation of phosphorylated PKA substrates demonstrated highly elevated phosphorylated Src in 3 of 4 tumors (Figure 6c). Furthermore, Tyr416 Src phosphorylation was elevated in two of four Prkar1aΔMam mammary tumors, suggesting that Src is a frequent mediator in PKA-initiated mammary tumors (Figure 6d). Together, these results highlight Src as a critical pathway impacted in breast tumors linked with aberrant PKA activity. PKA-triggered Src activation is responsible for mammary cell growth Next, the importance of Src in PKA-driven mammary epithelial cell transformation was dissected by evaluating cell proliferation and colony growth upon pathway modulation using inhibitors against ERα, ErbB2/EGFR, PI3K/Akt and/or Src. Prkar1aΔMam/PyMT mammary epithelial cells proliferated at a higher rate than PyMT, and cAMP promoted this phenotype (Figures 7a and b; Supplementary Figure 5A), as previously observed. Addition of the ERα antagonist fulvestrant or the ErbB2/EGFR inhibitor lapatinib had incomplete inhibitory effects on baseline and cAMP-induced proliferation in both PyMT and Prkar1aΔMam/PyMT genotypes (Figure 7b; Supplementary Figure 5A). In contrast, inhibition of Src with dasatinib reduced baseline, and profoundly prevented cAMPinduced proliferation of Prkar1aΔMam/PyMT mammary epithelial cells (Figures 7a and b). Wortmannin, which blocks PI3K/Akt, suppressed proliferation in a similar manner (Figures 7a and b). Mammary epithelial cell growth in Matrigel cultures provides a readout of transformation. Here, ERα inhibition by fulvestrant had no effect, whereas ErbB2/EGFR inhibition with lapatinib did not impact baseline Prkar1aΔMam/PyMT or cAMP-induced colony formation (Figures 7c and d; Supplementary Figure 5B). In contrast, both dasatinib and wortmannin significantly reduced baseline growth of colonies and additionally inhibited cAMPinduced colony formation in both genotypes (Figures 7c and d). To further dissect the importance of Src in Prkar1aΔMam/PyMT cell transformation, small interfering RNA (siRNA)-directed depletion of endogenous Src was performed. Four siRNAs (Src-si1, -si2, -si3 and -si4) targeting Src were transfected into Prkar1aΔMam/ PyMT mammary epithelial cells and Src knockdown was assayed 96 h post transfection by immunoblotting; all four siRNAs resulted in reduced Src protein levels, whereas a control siRNA had no effect (Figure 7e). Strikingly, Src depletion using two of the above siRNA duplexes (Src-si1 or Src-si4) significantly inhibited baseline and cAMP-stimulated Prkar1aΔMam/PyMT cell proliferation (Figure 7f; Supplementary Figure 5C) and 3D colony growth (Figure 7g). Thus, aberrant PKA activity in the mammary gland promotes epithelial cell proliferation and transformation through a mechanism involving the activation of the tyrosine kinase Src. Further, Src activation may act via Akt signaling to provide a growth advantage to these mammary epithelial cells. Poor patient survival in low-PRKAR1A/high-SRC human breast cancers Our findings that PKA hyperactivation drives mammary tumorigenesis in the mouse prompted us to examine this in human breast cancer. We first analyzed data from the TCGA data set34 and observed a significant correlation between altered PRKAR1A Oncogene (2014), 1 – 14

somatic copy number and expression (Pearson’s correlation coefficient, PCC = 0.67; P = 2.2 × 10
16; Supplementary Figure 6A). High PRKAR1A copy number/expression was associated with high expression of genes of the oxidative phosphorylation pathway, whereas low copy number/expression associated with high expression of genes of the ribosome pathway (Supplementary Figure 6B). Subsequent evaluation of PRKAR1A expression differences across tumor subtypes as defined using the PAM50 classifier indicated significant under-expression in the basal-like subtype relative to all others (t-test P-values o 0.001, Supplementary Figure 6C). Accordingly, PRKAR1A expression was positively correlated with ESR1, which encodes for ERα, and to a lower degree with ERBB2, which encodes for the human epidermal growth factor receptor 2 (HER2) (Supplementary Figure 6D). Our above experiments in the mouse had established increased Src signaling to be a main mechanism critical for PKA-directed mammary cell growth and transformation. To explore this molecular link in human breast cancer, the TCGA series was divided in two tumor sets corresponding to low or high PRKAR1A expression values (based on tertile categorization). Analyzing the expression differences between these sets identified an association with an oncogenic SRC signature previously derived in the non-tumorigenic MCF10A mammary cell line:35 genes overexpressed with SRC activation in MCF10A cells were also found to be over-expressed in breast tumors with low PRKAR1A expression (P = 0.023; Figure 8a, top left panel). This association was further confirmed by examining all genes over-expressed with SRC activation in MCF10A cells (at a false discovery rate o 5%); P = 0.003; Figure 8a, top right panel). The genes under-expressed with SRC oncogenic activation did not show significant results (Figure 8a, bottom panels). GSEA analysis in the TCGA data set revealed that low-PRKAR1A/high-SRC tumors are enriched for pathways regulating GPI anchor biosynthesis and the peroxisome metabolic pathways (Supplementary Figure 6E), while pathways regulating glycosphingolipid biosynthesis were under-expressed (Supplementary Figure 6F). Examining clinical characteristics in the TCGA tumors, those with concurrent low PRKAR1A and high SRC expression (low PRKAR1A/high SRC) were found to be more likely to recur (log-rank test P = 0.007; Figure 8b). In addition, recurrence was also higher in the low-PRKAR1A/high-SRC tumor subset compared with tumors classified as only low PRKAR1A, high SRC expression, or all others (log-rank test P-values o0.05; Figure 8c). Subsequently, examining the concordance with PAM50-based subtypes, 33% of the basallike and 17% of HER2 subtype had low-PRKAR1A/high-SRC expression (Figure 8d). Analysis of recurrence in these subtypes showed similar trends, although only HER2 reached significance despite containing fewer cases (P = 0.021; Supplementary Figure 6G). We repeated these analyses in the independent NKI-295 data set36 both for the full data and the HER2 subtype (Figures 8e and f; Supplementary Figure 6H). This recapitulated our findings from the TCGA analysis, showing similar trends when examining prognosis among tumors classified as low PRKAR1A/high SRC, low PRKAR1A, high SRC or others. Informed by our studies in mice, this work points to a distinct subset of human breast cancers with high rates of recurrence defined by low PRKAR1A/high SRC expression, and these tumors are found particularly within the basal-like and HER2 subtypes. DISCUSSION Here we describe a pro-neoplastic role for PKA isozyme hyperactivation in the mouse mammary gland. We show that mammary-specific loss of Prkar1a leads to elevated type-II PKA isozyme activation and this is sufficient to drive breast carcinogenesis. Further, we show that heightened PKA activity associates with Src pathway activation and this is in part responsible © 2014 Macmillan Publishers Limited

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* * * *

Figure 7. PKA hyperactivity induces proliferation through Src. (a) Mammary epithelial cell proliferation of Prkar1aΔMam/PyMT cells grown over 72 h. Cells were left untreated or stimulated with cAMP in combination with the small molecule inhibitors dasatinib (100 nM) or wortmannin (200 nM). Treatments were performed in triplicate and the experiments were replicated three times. (b) Comparison of cell proliferation at 72 h between Prkar1aΔMam/PyMT and PyMT cells treated with the small-molecule inhibitors fulvestrant, lapatinib, dasatinib or wortmannin. (c) The above cells were also subjected to Matrigel colony formation assays performed over 8 days. The images shown are representative of three independent experiments. (d) Small-molecule inhibitors dasatinib and wortmannin inhibit cAMP-induced colony formation in Prkar1aΔMam/ PyMT mammary epithelial cells, whereas fulvestrant and lapatinib do not. Bar graph represents number of cell colonies ⩾ 28 mm2 counted from indicated genotypes. (e) Immunoblot of Src from Prkar1aΔMam/PyMT mammary epithelial cells transfected with four siRNA duplexes directed against Src (Src-si1, -si2, -si3 and -si4), non-silencing siRNA control (NS) or transfection reagent alone (
) 96 h post transfection. (f) Cell proliferation over 96 h time course of Prkar1aΔMam/PyMT mammary epithelial cells transfected with NS, Src-si1 or Src-si4 siRNA. (g) Images of cAMP-stimulated (cAMP) or unstimulated (
) Prkar1aΔMam/PyMT mammary epithelial cell colonies at 5 days of culture following NS, Src-si1 or Src-si4 siRNA transfection. Quantification of colonies ⩾25mm2 in diameter are shown in the bar graph to the right where averages from five fields of view were calculated. Experiments were performed in triplicate on three independent occasions (n = 3). a = P ⩽ 0.05 compared with untreated control; b = P ⩽ 0.05 compared with cAMP-stimulated control; *P ⩽ 0.05 compared with NS. See also Supplementary Figure 5.

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Figure 8. Association between PRKAR1A/SRC expression levels, SRC oncogenic activation and breast cancer recurrence. (a) Gene set enrichment analysis (GSEA) graphical outputs for the association analysis of expression differences between high and low PRKAR1A-expressing TCGA tumors and genes over-/under-expressed with SRC oncogenic activation in MCF10A cells. The top panels correspond to the over-expressed genes in the original SRC oncogenic signature (left panel) or to the over-expressed genes at false discovery rate o5% (that is, SRC-mediated perturbation, right panel). Similarly, the bottom panels correspond to the under-expressed genes with SRC activation or mediated perturbation. The GSEA enrichment score and the nominal P-values are shown. (b) Kaplan–Meier breast cancer recurrence curves for categorization of TCGA tumors based on concurrent low PRKAR1A and high SRC expression versus the rest of cases with recurrence information available. The log-rank test P value and number of cases (n) are indicated. (c) Kaplan–Meier breast cancer recurrence curves for categorization of TCGA tumors based on low and/or high PRKAR1A/SRC expression values. (d) For the identification of low-PRKAR1A/high-SRC expression TCGA tumors, the complete series was ordered according to PRKAR1A or SRC expression and, subsequently, the intersection was identified. The distribution of these tumors (that is, low PRKAR1A plus high SRC) across the PAM50-based subtypes is shown in the right panel, which reveals higher concordance with basal-like type. (e) Kaplan–Meier survival curves for categorization of tumors based on concurrent low PRKAR1A and high SRC expression versus the rest of cases within each PAM50-based subtype in the full NKI-295 data set. (f) Kaplan–Meier survival curves for categorization of tumors based on low and/or high PRKAR1A/SRC expression values in the full NKI-295 data set. See also Supplementary Figure 6.

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11 for mammary epithelial cell proliferation and transformation. Our work additionally exposes a putative low-PRKAR1A/high-SRC human tumor subset associated with poor patient outcome. PKA is the main mediator of the highly conserved cAMP signaling pathway and aberrant activation of this kinase is known to be causal in hereditary endocrine neoplasias of the Carney complex.12,13 However, its importance in sporadic tumors originating from epithelial lineage is less understood. Mouse modeling of Prkar1a heterozygosity in the germ line setting recapitulates the spectrum of endocrine tumors seen in Carney complex patients harboring autosomal dominant inactivating PRKAR1A mutations.9 In mesenchymal cells, osteoblast-specific heterozygous Prkar1a deletion leads to spontaneous bone tumorigenesis.18,37 We now demonstrate that mammary epithelial cells lacking both copies of Prkar1a demonstrate aberrant type-II PKA activation. This mirrors reports of type-II PKA activity increases in Carney complex tumors.15,23 Importantly, we found that heightened PKA activation is responsible for highly augmented baseline and cAMP-induced proliferation of primary mammary cells in multiple assays in vitro and mammary lineage expansion in vivo, enabling stochastic mammary cancer development by 1 year of age. Although it is uncommon, breast ductal adenomas have been reported in women with Carney complex, indicating that this phenomenon is not limited to genetically engineered mice.38 Our findings generalize the importance of PKA holoenzyme homeostasis to epithelial tissue that impacts the most common form of cancer in women. A major finding of this study highlights the importance of typeII PKA as a critical mechanism in directing tumor cell proliferation. This observation is contrary to what has been previously reported about the roles of type-I and type-II PKA. Specifically, type-II PKA predominantly associates with well-differentiated non-proliferative cell types, while a strong interaction exists between type-I PKA isozyme, cell proliferation and tumorigenesis.8 A possible explanation for our findings centers on the complexity of PKA holoenzyme homeostasis, where the Prkar1a regulatory subunit exerts dominance over other regulatory subunits in regulating PKA activity. In our tumor model, where Prkar1a is absent in mammary epithelia, the increase in PKA activity can be attributed by either a compensatory increase in type-II PKA isozyme or by an increase in the unbound (unregulated) PKA catalytic subunit. In the MMTVPyMT background, hyperactive PKA activity (the result of increased type-II PKA or free PKA catalytic subunit) may cooperate with underlying PyMT cancer drivers (that is, Src) leading to accelerated tumorigenesis. The importance of Prkar1b in contributing to type-I PKA isozyme function cannot be overlooked in our mammary tumor model, even thought alterations in its expression were not significantly altered. It will be important to determine whether human breast cancers defined by low PRKAR1A exhibit an altered type-I/II PKA isozyme ratio. PKA has been studied as a mediator of ERα signaling in breast cancer, including ligand-dependent/-independent induction and tamoxifen resistance.2,5,29,39–41 Using small-molecule inhibitors, we ruled out the involvement of ERα and EGF receptor signaling in our PKA-driven mammary tumors. We also did not observe increases in expression of ERα-responsive genes Pgr or Greb1, or Wnt reporter genes Axin2 and Tcf4. Instead we found PKA-induced Src signaling to be a downstream mechanism in Prkar1aΔmam cohorts. It has been shown that the development of PyMT tumors depends in part on Src activity, where mammary epithelial ablation of Src significantly delays tumor onset.31 In our model, Prkar1aΔmam/PyMT tumors were associated with elevated phosphorylated Src at both serine 17 and tyrosine 416 residues. PKA-directed regulation of Src has previously been observed in fibroblasts and adrenal cells in vitro;33,42 our findings provide the first evidence of in vivo PKA-Src regulation. Although we did not directly examine Src activity in Prkar1aΔmam tumors, Src RNAi experiments demonstrated the importance of Src as a © 2014 Macmillan Publishers Limited

downstream mediator of PKA-directed cell proliferation and colony growth. Thus, mammary-specific PKA hyperactivation in this model cooperates with PyMT to augment Src-mediated proliferation and cellular transformation. Importantly, we observed Src activation even in the absence of the PyMT oncogene, highlighting the conserved signaling mechanism attributed to Prkar1a genetic ablation. However, our observation that not all Prkar1aΔmam tumors exhibited elevated phosphorylated Src (Y416) combined with our findings that Src inhibition partially blocked PKA-mediated cell proliferation suggests that aberrant PKA activity may interact with other unidentified signaling pathways important in tumorigenesis. Human breast cancers exhibit substantial complexity and integrative genomic studies are now progressively stratifying tumors into novel molecular subsets building on the PAM50 designations.19,20,34,43 These studies have exposed considerable heterogeneity within the major types of breast cancer. Patients with basal-like or HER2 breast cancers have adverse prognosis compared to those categorized as luminal A and luminal B. Aberrant Src activity has been found to correlate with HER2 positivity, higher breast tumor grade and inversely correlates with ERα status.16,17 Informed by the observation that Prkar1a deletion activates the Src pathway in the mouse, we probed for a putative low-PRKAR1A/high-SRC breast cancer subset and uncovered a substantial proportion of the patient population with tumors harboring this signature. In both the TCGA and NKI-295 breast cancer data sets, low-PRKAR1A/high-SRC tumors exhibited distinctly poor patient outcome, even when compared with low-PRKAR1A or high-SRC designations alone. Further, our analyses suggested that low-PRKAR1A/high-SRC tumors make up a notable proportion of basal-like as well as HER2 cancers. Given that SRC inhibitors such as dasatinib are in clinical trials for a variety of human cancers including advanced breast cancers, it will be important to determine whether SRC inhibitors can provide benefits to this putative novel low-PRKAR1A/high-SRC breast cancer subset. MATERIALS AND METHODS Reagents The mouse mammary epithelial cell line (NMuMG) was obtained from ATCC (CRL-1636). Ongoing cultures were maintained in DMEM containing 25 mM glucose, L-glutamine, antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin) and supplemented with 10% FBS. 8-bromoadenosine 3′, 5′-cyclic monophosphate (cAMP), cholera toxin, hydrocortisone, insulin and fulvestrant were purchased from Sigma Aldrich (St Louis, MO, USA). Phenol red-free, growth factor-reduced Matrigel was purchased from BD Biosciences (San Jose, CA, USA). EGF was purchased from Peprotech (Rock Hill, NJ, USA). Dasatinib and wortmannin were generously provided by Drs Shereen Ezzat and Vuk Stambolic, Ontario Cancer Institute, Toronto. Lapatinib was purchased from Selleck Chemicals. H89 was purchased from Cell Signaling Technology (Boston, MA, USA). PKI, InCELLect AKAP-StHt31inhibitor and InCELLect St-Ht31P control peptide were purchased from Promega (Sunnyvale, CA, USA). For cell signaling experiments, cells were washed in PBS and starved in DMEM media for 24 h; cells were treated with recombinant mouse EGF (10 nM) or insulin (50 nM) for 15 min. The following small molecules were used at the given concentrations: H89 (10 μM), PKI (20 μM), cAMP (1.0 mM), InCELLect AKAP-St-Ht31inhibitor and control peptide (5μM), fulvestrant (100 nM), lapatinib (100 nM), wortmannin (200 nM) and dasatinib (100 nM).

Mice MMTV-PyMT mice and MMTV-Cre mice in the FVB background were obtained from Dr W Muller (McGill University, Canada). The generation of the Prkar1alox/lox mice and genotyping of the WT, loxP and deletion allele have been described.16 To generate the experimental mice, the following breeding pairs were set: Prkar1alox/lox/MMTV-PYMT+/MMTV-Cre+ males were bred with Prkar1alox/lox/MMTV-PYMT–/MMTV-Cre
females. The proper Mendelian ratios were obtained. Mammary tumor onset was determined by manual palpation of mammary glands. Tumor endpoints, determined by Animal Care Committee guidelines, were reached when Oncogene (2014), 1 – 14

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12 mammary tumor diameter measured 1.5 cm. All animal experiments were approved by the Animal Care Committee, Ontario Cancer Institute.

Whole-mount analysis, immunohistochemistry and immunofluorescence Thoracic mammary glands from 40- or 90-day-old mice were analyzed for mammary morphology by carmine-alum whole-mount staining as described previously.44 For immunohistochemistry, 4% paraformaldehydefixed paraffin-embedded tissue sections were de-paraffinized in xylene, gradually rehydrated in descending concentrations of ethanol and subsequently treated in Borg Decloaker antigen retrieval solution (pH 9) for 30 min at 121 °C and 10 seconds at 90 °C using a Decloaking chamber (Biocare Medical, Concord, CA, USA). Tissue sections were stained using HRP-AEC tissue staining kit according to manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). For dual immunofluorescence microscopy, paraffin-imbedded sections were hydrated and subjected to antigen retrieval as described above. Tissues were permeabilized with 0.2% Tritin X-100 and 0.1% sodium borohydride, and blocked in 5% normal goat serum containing 0.1 mg/ml saponin. Primary antibodies (anti-keratin 14 and anti-kertain 18) diluted in PBS were incubated overnight at 4 °C in a humidified chamber. Primary antibodies were labeled with Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit secondary antibodies. Slides were mounted in DAPI mounting medium (Invitrogen, Carlsbad, CA, USA) and imaged using an Axio Observer inverted microscope (Carl Zeiss, Jena, Germany). Antibodies used were antimouse antibodies against keratin 14 (Covance, Princeton, NJ, USA), keratin 18 (Fitzgerald, Burlington, ON, Canada), Ki67 (Novus Biologicals, Oakville, ON, Canada), PR (hPRa7; generated in-house), ERα (clone M20, Santa Cruz Biotechnology; Dallas, TX, USA) and phosphorylated CREB (p-Creb Ser133) from Cell Signaling Technology (Beverly, MA, USA).

Mammary epithelial cell preparation, FACS analysis and cell sorting Single mammary cell suspensions were generated from freshly isolated pairs of fourth inguinal mammary glands of individual mice by enzymatic digestion and analyzed by flow cytometry as reported previously44 or cultured on collagen-coated tissue culture plates. Briefly, mammary glands were digested for 2.5 h at 37 °C in mouse Epicult-B with 5% FBS, 750 U/ml collagenase and 250 U/ml hyaluronidase. Organoids obtained after vortexing were subjected to red blood cell lysis in 0.8% NH4Cl, further dissociation in 0.25% trypsin for 2 min, 5 mg/ml dispase with 0.1 mg/ml DNase I for 2 min, and filtered through a 40-μm mesh to obtain single cells. All reagents were from StemCell Technologies Inc. (Vancouver, BC, Canada) and antibodies were from BD Pharmingen (San Diego, CA, USA) unless otherwise stated. Cells were blocked with Fc receptor antibody, incubated with biotinylated StemSep mouse/human chimera cocktail and anti-CD31 in order to label hematopoietic CD45+/Ter119+ cells and endothelial cells respectively, which were excluded by secondary conjugation with streptavidin-PE-Cy7 using flow cytometry. Dead cells were excluded from analysis by staining with propidium iodide (Sigma). Anti-CD49f-FITC (clone GoH3) and anti-CD24-R-PE (clone M1/69) were used to identify the mammary epithelial cell populations. FACS analysis was performed using FACSCalibur (BD) and FlowJo software (Tree Star Inc., Ashland, OR, USA). Cell sorting was performed on a FACSAria (BD). The purity of sorted populations was routinely greater than 96%.

RNA isolation and real-time PCR analysis Total RNA was prepared from FACS-sorted primary mammary cell subpopulations using the PicoPure RNA Isolation Kit (Arcturus, Carlsbad, CA, USA) as described in Joshi et al.44 The quality and concentration of RNA was determined by visualizing purified RNA samples on SyBr Green II (Invitrogen)-stained formaldehyde agarose gels and by analysis with a NanoDrop 2000 Spectrometer (260/280 ratio; Thermo Scientific, Waltham, MA, USA). Isolated and purified total RNA was reverse transcribed into first strand cDNA and amplified using the SMARTer PCR cDNA Synthesis Kit and Advantage2 PCR Kit (Clontech). Amplified cDNA aliquots (5 μl) were analyzed on ethidium bromide-stained agarose gels (1.2%) to determine the optimal number of LD-PCR cycles for cDNA amplification that ranged from 18–21 cycles. Relative quantification real-time PCR (ΔΔCt) was performed on 4 ng of cDNA generated from FACS-purified primary mammary cells using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). TaqMan gene expression assay Oncogene (2014), 1 – 14

mix containing unlabeled PCR primers and FAM labeled TaqMan MGB probes were used to detect expression of specific genes as listed by the catalog numbers in Supplementary Table 1. All raw data were analyzed using Sequence Detection System software Version 2.1 (Applied Biosystems). The threshold cycle (CT) values were used to calculate relative RNA expression levels. Expression levels of target genes were normalized to endogenous β-actin transcripts and compared with control MMTV-Cre or PyMT luminal population (relative expression = 1).

Immunoprecipitation and immunoblot analysis Protein (500 μg) from whole mammary gland extracted by RIPA extraction buffer (Tris–HCl pH 7.6, 1% Triton X-100, 0.1% SDS, 1% NP-40, 1% sodium deoxycholate, 5 mM EDTA, 50 mM NaCl) supplemented with 200 μM Na3VO4, 200 μM NaF, 2 mM PMSF and an appropriate dilution of Complete Mini, EDTA-free protease inhibition cocktail tablets (Roche, Indianapolis, IN, USA) was subjected to immunoprecipitation overnight at 4 °C using (1) an antibody that specifically recognizes RRXS*/T*-phosphorylated PKA substrate epitopes (Cell Signaling Technology) or (2) a polyclonal antibody recognizing the R2β PKA regulatory subunit (ab38949, Abcam, Cambridge, UK). Immunoprecipitated protein was purified following incubation with protein G plus protein A/G agarose beads (Invitrogen) for 1 h. The precipitated protein complexes were washed at 4 °C in RIPA buffer lacking sodium deoxycholate and SDS and was then subjected to SDS–PAGE followed by immunoblotting. For immunoblotting, cells were washed in PBS and incubated in RIPA cell extraction buffer, for 30 min. Protein concentrations were determined using a BCA kit (Pierce Chemicals; Rockford, IL, USA). Cell protein lysate (30 μg) was resolved by SDS–PAGE and transferred to nitrocellulose membranes. The membranes were probed using the following mouse antibodies: anti-AKAP1, antiphosphorylated CREB (pCreb, Ser133) anti-Creb, Akt/PKB (p-Akt, Ser473), anti-Akt/PKB, anti-phosphorylated Src (Ser17), anti-phosphorylated Src (Tyr416), anti-Src (all from Cell Signaling Technology). The blots were stripped and re-probed with an HRP-conjugated monoclonal antibody directed against mouse β-actin (Santa Cruz Biotechnology).

siRNA transfection Four ON-TARGETplus siRNAs (Thermo Scientific; LQ-040877-00-0002; 25 nM) targeting the mouse c-Src mRNA transcript (Src-si1 50 -CCAAGGGC CUCAACGUGAA-30 ; Src-si2 50 -CCUCAGGCAUGGCGUACGU-30 ; Src-si3 50 -CGUCCAAGCCGCAGACUCA-30 ; Src-si4 50 -GAGAACCUGGUGUGCAAAG-30 ) were transfected into primary mammary epithelial cells using Lipofectamine RNAi Max transfection reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Cells transfected with ONTARGETplus Non-silencing siRNA#1 (NS; Cat# D-001810-01-20) or cultured in the presence of transfection reagent alone, served as negative controls.

Mammary cell proliferation assays Cells were seeded at 2 × 103 cells/well into opaque collagen-coated 96-well microplates in 100 μl of DMEM/F12 1:1 medium containing 2% charcoalstripped FBS, EGF (5 ng/ml), insulin (10 ng/ml), LA complex (5 μg/ml), penicillin/streptomycin and anti-microbiotic (Wisent, St Bruno, QC, Canada). Cells were cultured for 0, 24, 48, 72 or 96 h in the presence of cAMP (1.0 mM) or the small molecules H89, PKI, fulvestrant, lapatinib, wortmannin or dasatinib, after which cell proliferation was measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) following the manufacturer’s instructions on a POLARstar Omega plate reader (BMG; Offenburg, Germany). In vitro Matrigel colony forming assays were performed as described45 with modifications. Single mammary cell suspensions were prepared from individual mice, and 5000 total mammary cells from the inguinal glands of PyMT or Prkar1aΔMam /PyMT were seeded onto 80 μL of pre-coated Matrigel in eight-well chamber slides and cultured in DMEM/F12 1:1 medium containing 2% charcoal-stripped FBS, 2.5% Matrigel, EGF, insulin and cholera toxin (200 ng/ml). After 24 h, culture media was replaced with media containing the small-molecule inhibitors described above. All untreated cells were supplemented with 1.0% DMSO. Cell colonies were fixed at day 8 of culture in 4% paraformaldehyde for 30 min at 4 °C. Colonies were imaged using an inverted light microscope using a × 4 objective; colonies were counted in five random fields of view. Colony size (μm2) was determined by measuring the height and width of each colony using Image J Software. © 2014 Macmillan Publishers Limited

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FPLC chromatography

ACKNOWLEDGEMENTS

All procedures were performed at 4 °C. Samples (1.5 mg protein in 500 μl PKA protein buffer) were separated using a 5 ml Bio-Scale Mini Macro-Prep DEAE cartridge (Bio-Rad, Mississauga, ON, Canada) on an Akta FPLC System (GE Healthcare, Buckinghamshire, UK) at a flow rate of 1.0 ml/min. Samples were loaded onto the column in low-salt buffer (10 mM Tris–HCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.1). The column was washed with 25 ml of low-salt buffer (five column volumes), collecting 5-ml fractions. The column was then eluted with a linear NaCl gradient (0.0–0.8 M) from low-salt buffer to high-salt buffer (10 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol, 1.0 M NaCl, pH 7.1) over 25 ml (five column volumes), collecting 0.5-ml fractions. The column was cleaned with 25 ml (five column volumes) of high-salt buffer before re-equilibrating to low-salt buffer for the following run. A total of 100 μl of each fraction was precipitated with 200 μl trichloroacetic acid (50%) at 4 °C for 20 min. The precipitated fractions were centrifuged at 14 000 × g, 15 min, 4 °C. Supernatants were discarded, the pellets were washed with 500 μl cold acetone and centrifuged again. The acetone wash was removed, the pellets were air-dried and resuspended in Laemmli buffer (Tris–HCl 375 mM, pH 6.8; SDS 9%; glycerol 50%; betamercaptoethanol 9%; bromophenol blue 0.03%) where samples were resolved by PAGE followed by immunoblotting using antibodies directed against R1α (BD Transduction Labs, Lexington, KY, USA) and R2β (BD Transduction Labs). The remaining 400 μl of each fraction was concentrated down to 40 μl using 10 -kDa-cuttoff Microcon Centrifugal filters (Millipore, Billerica, MA, USA). These concentrated protein samples where then used to determine PKA activities (10 μg/reaction).

We thank Megan K Barker and Paul Waterhouse for critical reading of the manuscript. We would also like to acknowledge Shareen Ezzat for sharing the small molecule Dasatinib, Vuk Stambolic for offering insight into biochemistry experiments and the IDIBELL’s Biostatistics Unit for help in analyzing breast cancer data sets. This work was supported by a Canadian Breast Cancer Foundation (CBCF) grant to RK and the Spanish Ministry of Health grant FIS-PI12/01528 and RD12/0036/0008 to MAP. AGB holds a CBCF fellowship.

REFERENCES

The authors declare no conflict of interest.

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PKA activity assay The SignaTech protein kinase (PKA) assay system (Promega) was used to measure basal (untreated) and total (cAMP-treated) PKA activities from total mammary gland protein lysates, FACS-purified mammary epithelial cells and concentrated FPLC protein fractions. Briefly, 10 μg of protein lysate containing 32P-labeled phosphate was incubated with biotinylated Kemptide, a PKA-specific substrate, in the absence or presence of 0.01 mM cAMP. PKA activity specificity was determined using the PKA inhibitor PKI (20 μM). The 32P-labeled biotinylated substrate is recovered from the reaction mix using perforated SAM2 Biotin Capture Membranes that bind to and immobilize the substrate. PKA activity is then measured using standard scintillation to determine radioactive counts/min, reflecting the amount of phosphorylated substrate. All determinations of PKA activity were performed in duplicate, corrected for protein concentration, and an average value was calculated for each experiment.

Bioinformatics The publicly available TCGA data were downloaded from the corresponding repository (tcga-data.nci.nih.gov/tcga/tcgaDownload) on July 2012. This set contained expression data for 509 primary tumors, of which 241 have also recurrence information available. The TCGA-normalized and preprocessed data were used in subsequent analyses. The PAM50 predictor was applied as previously described.46 Of the 241 tumors set, 65 were classified as basal-like, 30 as HER2, 73 as luminal A, 57 as luminal B and 16 as normal-like. The raw MCF10A expression data were downloaded from the Gene Expression Omnibus reference GSE3151. Data were normalized and the significance analysis of microarrays (SAM) algorithm47 was applied to identify differentially expressed probes at a ⩽ 5% false discovery rate (over-expressed, n = 1600; under-expressed, n = 261). Gene expression and clinical data for the NKI data set were downloaded from the Stanford Microrray Database. The PRKAR1A and SRC genes were represented by a single probe each in the NKI data set (NM_002734 and NM_005417, respectively). Correlation, clustering and survival analyses were carried out using R/Bioconductor software (Seattle, WA, USA). The GSEA tool48 was run using default values for all parameters.

Statistics Data are reported as mean ± standard error of the mean. All calculations were carried out using GraphPad Prism software (La Jolla, CA, USA). Comparisons were made by two-tailed Student’s t-test and ANOVA. The differences were accepted as significant at Po0.05.

CONFLICT OF INTEREST

PKA-induced Src drives mammary tumorigenesis AG Beristain et al

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