(214), ra19. [DOI: 10.1126/scisignal.2001986] 5Science SignalingMillard, Gordon B. Mills, Joan S. Brugge and John G. Albeck (6 March 2012) Devin T. Worster, Tobias Schmelzle, Nicole L. Solimini, Eric S. Lightcap, Bjorn

Kip2p57Cells to the Growth Factors IGF-1 and EGF Through the Cell Cycle Inhibitor

Akt and ERK Control the Proliferative Response of Mammary Epithelial`

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R E S E A R C H A R T I C L E

C A N C E R

Akt and ERK Control the Proliferative Response ofMammary Epithelial Cells to the Growth FactorsIGF-1 andEGFThrough theCellCycle Inhibitorp57Kip2

Devin T. Worster,1 Tobias Schmelzle,1* Nicole L. Solimini,1† Eric S. Lightcap,2 Bjorn Millard,3

Gordon B. Mills,4 Joan S. Brugge,1‡ John G. Albeck1

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Epithelial cells respond to growth factors including epidermal growth factor (EGF), insulin-like growthfactor 1 (IGF-1), and insulin. Using high-content immunofluorescence microscopy, we quantitated differ-ences in signaling networks downstream of EGF, which stimulated proliferation of mammary epithelialcells, and insulin or IGF-1, which enhanced the proliferative response to EGF but did not stimulate prolif-eration independently. We found that the abundance of the cyclin-dependent kinase inhibitors p21Cip1 andp57Kip2 increased in response to IGF-1 or insulin but decreased in response to EGF. Depletion of p57Kip2,but not p21Cip1, rendered IGF-1 or insulin sufficient to induce cellular proliferation in the absence of EGF.Signaling through the PI3K (phosphatidylinositol 3-kinase)–Akt–mTOR (mammalian target of rapamycin)pathway was necessary and sufficient for the increase in p57Kip2, whereas MEK [mitogen-activated or ex-tracellular signal–regulated protein kinase (ERK) kinase]–ERK activity suppressed this increase, forming aregulatory circuit that limited proliferation in response to unaccompanied Akt activity. Knockdown ofp57Kip2 enhanced the proliferative phenotype induced by tumor-associated PI3K mutant variants and re-leased mammary epithelial acini from growth arrest during morphogenesis in three-dimensional culture.These results provide a potential explanation for the context-dependent proliferative activities of insulinand IGF-1 and for the finding that the CDKN1C locus encoding p57Kip2 is silenced in many breast cancers,which frequently show hyperactivation of the PI3K pathway. The status of p57Kip2 may thus be an impor-tant factor to assess when considering targeted therapy against the ERK or PI3K pathways.

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INTRODUCTION

In mammalian cells, extracellular growth factor–mediated signaling,which is essential for cellular proliferation, is frequently disrupted incancer. Activation of growth factor receptors leads to the stimulationof numerous downstream pathways that modulate cellular metabolism,control gene transcription, and engage the cell cycle. Of these pathways,the Ras-Raf-ERK (extracellular signal–regulated kinase) and PI3K(phosphatidylinositol 3-kinase)–Akt pathways play a central role in driv-ing many of the phenotypic changes induced by growth factors. TheRas-Raf-ERK pathway (hereafter the ERK pathway) is typically stimu-lated by recruitment of guanine nucleotide exchange factors such as SOS(son of sevenless) to the growth factor receptor, inducing the small gua-nosine triphosphatase Ras to bind guanosine triphosphate (GTP). Afterthis activation step, Ras binds to and activates the kinase Raf, which stim-ulates a kinase cascade culminating in the phosphorylation of ERK byMEK (mitogen-activated or extracellular signal–regulated protein kinasekinase). Phosphoactivation of ERK results in its nuclear translocationand phosphorylation of numerous substrates to both promote proliferation

1Department of Cell Biology, Harvard Medical School, Boston, MA 02115,USA. 2Millennium Pharmaceuticals, Cambridge, MA 02139, USA. 3De-partment of Systems Biology, Harvard Medical School, Boston, MA 02115,USA. 4Department of Systems Biology, University of Texas M. D. AndersonCancer Center, Houston, TX 77030, USA.*Present Address: Novartis Institutes for Biomedical Research, Disease AreaOncology, CH-4002 Basel, Switzerland.†Present Address: Department of Genetics, Harvard University MedicalSchool and Division of Genetics, Brigham and Women’s Hospital, Boston,MA 02115, USA.‡To whom correspondence should be addressed. E-mail: joan_brugge@hms.harvard.edu

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and inhibit proapoptotic signals (1). The PI3K-Akt pathway (hereafter theAkt pathway) is activated by binding of the PI3K p85 regulatory subunitto tyrosine-phosphorylated sites on growth factor receptors or on asso-ciated adaptor proteins such as IRS-1 (insulin receptor substrate 1) orGab1. This binding leads to activation of the associated p110 catalytic do-main, which phosphorylates phosphatidylinositol 4,5-bisphosphate in theplasma membrane to generate phosphatidylinositol 3,4,5-trisphosphate(PIP3). Akt contains a pleckstrin homology (PH) domain, which bindsto PIP3, enabling recruitment of Akt to the membrane, where it is phos-phorylated and activated by another PH domain–containing protein ki-nase, PDK1 (phosphoinositide-dependent protein kinase 1), and by theTORC2 [target of rapamycin (TOR) complex 2] signaling complex. Asa downstream effector of PI3K signaling, Akt promotes protein synthesis,cell proliferation, cell metabolism, and cell survival (2).

The ERK and Akt pathways work coordinately or synergistically topromote cell growth and progression through the cell cycle. Both path-ways are involved in the activation of the kinase mTOR (mammalianTOR, which controls the rate of cellular growth by promoting proteintranslation) (3), c-Myc (a key transcription factor controlling both metab-olism and cell cycle progression) (4), and cyclin D (a central regulator ofthe transition from G1 to S phase of the cell cycle) (5, 6). Other down-stream effectors are specific to one of the two pathways; for example, theAkt pathway stimulates the uptake of glucose (7), whereas ERK activatesimmediate-early transcription factors including c-Fos and Ets-1. Becausethe ERK and Akt pathways are pleiotropic and interconnected, it is diffi-cult to determine the distinct contribution of each to the overall prolifera-tive response to growth factors. Nonetheless, both phosphorylated Akt andERK (pAkt and pERK, respectively) are frequently used as readouts ofproliferative or oncogenic signaling.

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The ERK and Akt pathways also engage in negative crosstalk. For ex-ample, Akt suppresses ERK pathway signaling through inhibition of Raf(8–10). Moreover, ERK and its target p90RSK (p90 ribosomal S6 kinase)contribute to mTOR activation through phosphorylation of the tuberoussclerosis complex (TSC1/2), and the downstream mTOR effector S6 ki-nase 1 (S6K1) stimulates a negative feedback loop, thereby suppressingAkt activity (3). Thus, negative crosstalk could maintain a proper balancebetween the outputs of each pathway, preventing hyperactivation of asingle pathway that might lead to uncontrolled proliferation. However,how the dynamic interplay between these pathways controls proliferation,what combinations of signals constitute pathological or deregulated states,and what mechanisms are used by the cell to detect and suppress poten-tially oncogenic signaling states remain to be fully elucidated.

Also central to cell cycle control are the cyclin-dependent kinase(CDK) inhibitors, which fall into two families: INK4 (inhibitor of kinase4) and CIP/KIP (CDK interacting protein/kinase inhibitory protein). TheCIP/KIP family, consisting of CDKN1A, CDKN1B, and CDKN1C(p21Cip1, p27Kip1, and p57Kip2, respectively, hereafter referred to as p21,p27, and p57), bind to multiple cyclin-CDK complexes and can arrest thecell cycle during G1, S, or G2 phases. These proteins may also play a crit-ical role in cell cycle progression by acting as assembly factors for theCDK4/6–cyclin D complex, promoting late G1 to S phase transition(11). Within the context of growth factor–mediated signaling, p27 is rec-ognized as a central regulator of the balance between quiescence and pro-liferation (12). Akt phosphorylates p27, inducing its translocation to thecytoplasm, where it is degraded (13). p21 is best known for its role in p53-mediated cell cycle arrest in response to DNA damage or other stresses(14, 15) and has been reported to be either stimulated or suppressed byAkt (16, 17) and potentially activated by the ERK pathway (18). Althoughless is known about p57, it is the only member of the CIP/KIP familyessential in mouse embryonic development (19). p57 has tumor suppres-sor functions, being silenced in many cancers and implicated in Beckwith-Wiedemann syndrome, a congenital disorder associated with tissueovergrowth and an increased risk of cancer (20). At the mRNA level,p57 is stimulated by glucocorticoids (21) and suppressed by the Myc-induced microRNA-221/222 (miR-221/222) (22, 23).

Here, we have identified p57 as an effector of crosstalk between theERK and the Akt pathways; activation of the Akt pathway increased theabundance of p57, whereas the ERK pathway suppressed it. This cross-regulatory motif played a key role in determining the proliferative re-sponse of mammary epithelial cells to epidermal growth factor (EGF)and insulin-like growth factor 1 (IGF-1) or insulin. Activation of thePI3K pathway in the absence of ERK activity, either by IGF-1 or insulinor by mutant PI3K isoforms, led to an increase in the abundance of p57and proliferative arrest. Depletion of p57 enabled IGF-1–induced prolif-eration in the absence of EGF, enhanced proliferation of cells harboringendogenous PI3K mutations, and abrogated proliferative arrest in three-dimensional (3D) culture. Thus, regulation of p57 by ERK and Akt acts asa network sensor capable of detecting and limiting the proliferative re-sponse to “imbalanced” signaling states in which the Akt pathway is acti-vated in isolation.

RESULTS

Growth factor–mediated changes in p57 abundancecontrol proliferationMCF-10A mammary epithelial cells are typically cultured in growth me-dium containing both insulin and EGF (with insulin present at a concen-tration capable of stimulating both the insulin and the IGF-1 receptors). To

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examine the contribution of these mitogens to proliferative behavior, wecultured a clonal derivative of MCF-10A cells in the presence of variousconcentrations of EGF and either insulin or IGF-1 and assessed prolifer-ation by high-content immunofluorescence microscopy (HCIF). We usedphosphorylated retinoblastoma protein (pRb) as an indicator of cell cycleprogression (24). A small percentage of cells (5%) were pRb-positive afterdeprivation of both EGF and insulin for 24 hours, whereas the greatestamount of pRb staining (81%) was observed at maximal concentrationsof EGF in the presence of IGF-1 or insulin (Fig. 1A). Although EGF alonewas capable of stimulating cell proliferation in a dose-dependent manner,concentrations of insulin and IGF-1 capable of fully activating the Akt path-way (25) had no effect. At submaximal concentrations of EGF (0.5 ng/ml),costimulation with IGF-1 or insulin displayed a cooperative effect, increas-ing the pRb-positive population from 22 to 45 or 35%, respectively. Thus,both EGF and IGF-1 induced proliferative signals, but those downstreamof IGF-1 required additional input from the EGF-stimulated network tobe manifest.

In agreement with previous work (26), EGF potently stimulated phos-phorylation of ERK, but stimulated the Akt pathway relatively weakly, asindicated by a modest shift (from 1.18 to 1.14) in the nuclear/cytoplasmicratio of FoxO3a (a more sensitive indicator of Akt activity than pAkt itselfin MCF-10A cells; Fig. 1B). In contrast, IGF-1 induced stronger cyto-plasmic localization of FoxO3a (nuclear/cytoplasmic ratio = 1.04), indicat-ing high PI3K-Akt activity, but did not induce detectable phosphorylationof ERK.

To examine the signals contributing to proliferation downstream ofEGF and IGF-1, we measured a panel of 12 proliferation-related signalingproteins by HCIF (Fig. 1C). This quantitative analysis confirmed the dif-ferences between EGF and IGF-1 in activation of ERK and Akt signaling.As expected, the abundance or phosphorylation of numerous proteins thatsignal downstream of both ERK and Akt, including c-Myc, c-Fos, andpS6, changed in response to both EGF and IGF-1. For most of theseco-regulated proteins, induction by EGF was stronger than by IGF-1, sug-gesting that the differences in proliferative response between EGF andIGF-1 could in part be due to stronger activation of ERK effectors, includ-ing c-Fos and c-Myc. However, abundance of the cell cycle regulators p21and p57 increased specifically in response to IGF-1, but not EGF, suggest-ing that these proteins may contribute to the differential phenotypic re-sponse to the two growth factors.

A more detailed analysis of p57 dynamics revealed that saturating con-centrations of IGF-1 increased the median p57 abundance by about two-fold (Fig. 2, A and B). Costimulation with EGF blocked this increase in adose-dependent manner; IGF-1 up-regulation of p57 was fully blocked byconcentrations of EGF of 2 ng/ml or higher. EGF also suppressed the basal(non–IGF-stimulated) p57 expression. Changes in p21 abundance fol-lowed a similar trend, but were less pronounced and more prone to exper-imental variability (Fig. 2C and fig. S1). Covariate analysis of single-celldata for p57 in combination with pRb revealed that p57 was present pri-marily in pRb-negative cells. When the frequency of pRb-positive cellswas evaluated as a function of p57 abundance by means of a sliding win-dow, the percent of pRb-positive cells decreased markedly as p57 abun-dance increased (Fig. 2D). This relationship, which was consistent acrossall treatment conditions (fig. S2), enabled us to define a threshold repre-senting the critical amount of p57 required for its cell cycle–inhibitoryeffect. The frequency of cells in which p57 was above this critical thresh-old (p57-positive cells) increased from 27% in untreated cells to 57% inIGF-1–treated cells (Fig. 2E). Cotreatment with EGF (20 ng/ml) reducedthe frequency of p57-positive cells to 8%. Thus, IGF-1 and EGF inducechanges in p57 abundance that are quantitatively relevant to the control ofcell cycle progression.

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Covariate analysis of p21 and pRb revealed a relationship similar tothat seen with p57 (Fig. 2D). However, the range in percentage of p21-positive cells across all conditions was smaller than that observed with p57(Fig. 2E; 24 to 55% for p21 compared to 8 to 57% for p57). Moreover, theabundances of p21 and p57 were not correlated at the single-cell level(Fig. 2F). This relationship supports the hypothesis that these CDK inhib-itors act independently to limit proliferation. The quantitative difference inresponse between these CDK inhibitors argues that p57 plays a larger rolein controlling the overall proliferative rate of the population in response toIGF-1 and EGF stimulation.

Accordingly, we next asked whether activation of p21 or p57 was in-volved in suppressing cell proliferation in response to IGF-1. We depletedp57 in MCF-10A cells with four unique short hairpin RNA (shRNA)constructs (Fig. 3, A and C, and fig. S3). Relative to cells transduced witha control shRNAvector or uninfected cells, cells expressing shRNAs tar-geting p57 displayed a 1.5- to 2-fold increase in proliferative responseto IGF-1 (Fig. 3D). This increase in proliferation rate was more subtlethan that produced by saturating concentrations of EGF, but nonethelessled to a significant (P < 0.001) increase in cell number, as confirmed ingrowth curve experiments (Fig. 3E). In contrast, no such enhancement ofIGF-1–stimulated proliferation was observed in p21−/− MCF-10A cells(Fig. 3F) (27). Thus, p57, but not p21, appears to limit IGF-1– or insulin-induced proliferation. We therefore focused on the mechanisms involvedin IGF-1– and EGF-dependent regulation of p57.

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The Akt pathway increases p57 abundanceBecause insulin activated the Akt pathway more strongly than did EGF,we examined whether this pathway was involved in stimulating the in-crease in p57 abundance. Small-molecule inhibitors of PI3K activity(GDC-0941), mTOR kinase activity (Torin-1), or both (BEZ-235) attenu-ated IGF-1–mediated p57 up-regulation (Fig. 4, A and B). Of these inhib-itors, Torin-1 was most effective in reducing the frequency of p57-positivecells [to 5%, relative to 62% for the dimethyl sulfoxide (DMSO) control]followed by BEZ-235 (10%) and GDC-0941 (22%). Rapamycin, whichspecifically targets mTORC1, had a modest effect, reducing the frequencyof p57-positive cells to 40%. The efficacy of inhibitors capable ofblocking both mTORC1 and mTORC2 (Torin-1 and BEZ-235), togetherwith the weaker effect of rapamycin, suggests that the IGF-1–stimulatedincrease in p57 abundance is driven by mTORC2 or a combination ofmTORC1 and mTORC2.

To determine whether Akt pathway activation alone was sufficient toincrease p57 abundance, we expressed an inducible myristoylated Aktvariant (myrAkt-ER) that can be selectively activated by treatment with4′-hydroxytamoxifen (4OHT) (28) in MCF-10A cells. Cells expressingmyrAkt-ER were first cultured for 48 hours in the absence of growthfactors to suppress all other proliferative signals and then stimulated with4OHT at various time points before fixation. Treatment with 4OHT aloneinduced a marked increase in p57, whereas cotreatment with EGF attenu-ated the 4OHT-induced p57 response (Fig. 4C). Temporal analysis revealed

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Fig. 1. Differential regulation of proliferationand the Akt and ERKpathways by EGF, insulin, and IGF-1. (A) Percentage of pRb-positivecells at various EGFconcentrations in theabsence or presenceof insulin (10 mg/ml) oIGF-1 (100 ng/ml). pRbwas detected by HCIFafter 24 hours. Valuesindicate the means ±SD of triplicate measurements from onerepresentative experiment that was repeated three times**P<0.01. (B) Immunofluorescence imagesof pERK, FoxO3a, andpRb in response toEGF (20 ng/ml) or IGF-1(100 ng/ml). Numbersin the lower right indicate the average ratioof nuclear to cytoplasmic staining intensity

for FoxO3a. Images are representative of three independent ex-periments. (C) Quantitation of key signaling proteins in cells treated withEGF (20 ng/ml) or IGF-1 (100 ng/ml). The indicated protein signals weredetected by HCIF after 24 hours. Histograms represent distributions

of single-cell intensities, where the x axis represents the intensity othe signal, and the y axis represents the frequency of cells at each intensity normalized with the mode as 1. Data represent two independenexperiments.

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that the induction of p57 began 4 hours after treatment with 4OHT andincreased steadily through the 24-hour time period (Fig. 4D). However, noproliferative response, as assessed by pRb staining, was observed after4OHT treatment (fig. S4). Therefore, activation of Akt is sufficient to in-crease the abundance of p57, a response that can be suppressed by anEGF-stimulated pathway.

The ERK pathway suppresses p57Because EGF is a potent activator of the ERK signaling pathway, we as-sessed the involvement of ERK activity in suppressing p57. Treatment ofEGF- or EGF + IGF-1–stimulated cells with a MEK inhibitor (PD0325901)led to a marked increase in the frequency of p57-positive cells (from 6 to56% in EGF-treated cells and from 9 to 87% in EGF + IGF-1–treatedcells; Fig. 5, A and B). In contrast, MEK inhibition in unstimulated orIGF-stimulated cells did not substantially increase the abundance ofp57, in accord with the lack of detectable ERK activity under theseconditions. Additionally, overexpression of H-RasV12, which constitutivelystimulates ERK activity, resulted in nearly complete suppression ofIGF-1–stimulated p57 induction (Fig. 5C). Treatment of H-RasV12–expressing cells with a MEK inhibitor reversed this suppression ofp57, confirming the requirement for ERK-mediated signaling.

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To elucidate the relationship between the ERK and the Akt pathwaysin controlling p57 abundance, we examined the effects of inhibiting bothpathways simultaneously. Under conditions of EGF stimulation, the in-crease in p57 manifest with MEK inhibition was effectively blocked byTorin-1 or BEZ-235 (Fig. 5D), indicating that PI3K and mTOR activityinduced by EGF is sufficient to up-regulate p57, but is typically blockedby concomitant activation of the ERK pathway. Additionally, we plottedp57 abundance as a function of that of pERK and pAkt for all conditionsshown in Figs. 2A, 4A, and 5A (including varying concentrations of EGF,insulin, IGF-1, MEK inhibitors, and PI3K-TOR inhibitors) (Fig. 5E). Fromthis plot, it is apparent that under conditions of low ERK activity, p57 abun-dance is proportional to that of pAkt. However, p57 abundance is very lowwith increased pERK, regardless of the amount of pAkt. Thus, p57 accu-mulates in response to Akt signaling, whereas ERK acts as a dominant sup-pressor of this accumulation.

Western blotting confirmed that the IGF-1–PI3K– and EGF-ERK–mediated effects on p57 immunofluorescence reflected changes in totalcellular p57 protein (fig. S5). To assess whether these effects on p57 abun-dance occur at the transcriptional level, we measured p57 mRNA abun-dance by quantitative real-time polymerase chain reaction (qPCR).Although insulin stimulated a modest (1.2-fold) increase in p57 mRNA

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Fig. 2. Dynamics of p57 and p21 regulation coordinated by IGF-1 and EGF.(A) HCIF images of p57 in response to varying combinations of EGF andIGF-1. MCF-10A cells were treated as in Fig. 1. (B and C) Histograms of p57(B) or p21 (C) abundance determined by HCIF for the conditions shown in(A). a.u., arbitrary units. (D) Covariate single-cell analysis of p21 or p57 andpRb. Top: density scatter plots of p57 or p21 versus pRb signal for individ-ual cells as measured by HCIF. Bottom: analysis of pRb as a function of p57or p21 for cells treated with EGF (0.5 ng/ml) and IGF-1 (100 ng/ml). Individ-ual cell measurements were binned according to p57 or p21 abundance

(x axis), and the percentage of pRb-positive (% pRb+) cells present in eachbin was calculated. Curves indicate the mean, and points the individualvalues, of the % pRb+ for each bin from triplicate measurements. Verticalpink lines denote thresholds for p57 or p21 abundance determined by themidpoint of the decline in % pRb+. (E) Frequency of p57- or p21-positivecells under different growth factor conditions, using the thresholds definedin (D). (F) Covariate analysis of p21 and p57 abundance for cells treatedwith EGF (0.5 ng/ml) and IGF-1 (100 ng/ml). Data are from individualexperiments representative of at least three independent replicates.

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abundance, treatment with the dual PI3K and mTORinhibitor PIK-90 reduced p57 mRNA abundanceby ~2-fold (Fig. 5F). In contrast, insulin-stimulatedp57 mRNA abundance was reduced 3-fold by EGFtreatment, and this effect was fully blocked by theMEK inhibitor PD98059. Additionally, under di-rect stimulation of Akt signaling with the myrAkt-ER variant, p57 mRNA abundance was increased~4.3-fold above the control (Fig. 5G). These changesin p57 mRNA abundance closely resemble the reg-ulation detected by HCIF (Figs. 2, 4, and 5), sug-gesting that the changes in p57 observed here occurmainly at the level of mRNA abundance.

To establish the timing of changes in p57mRNA in response to growth factor signaling,we performed time course measurements of p57mRNA upon EGF treatment or withdrawal. Whencells growing in the presence of IGF-1 alone werecotreated with EGF, p57 mRNA abundance de-creased rapidly between 3 and 6 hours (Fig. 5H).When MCF-10A cells growing in the presence ofEGF and IGF-1 were shifted to IGF-1 alone, anincrease in the abundance of p57 mRNAwas ap-parent by 2 hours and reached a plateau by 12 hours(Fig. 5I). These kinetics are consistent with thetime scales of transcriptional induction by growthfactor signaling pathways and mRNA turnover(29, 30).

The ERK and Akt pathways controlp57 in epithelial and tumor cellsTo determine the range of cell types in which Akt-and ERK-dependent regulation of p57 is relevant,we assessed the function of this network in vari-ous epithelial, nonepithelial, and cancer cell lines.With MCF-12A and 184A1 cells, nontransformedmammary epithelial lines derived from different in-dividuals, p57 responses to IGF-1 and EGF stimu-lation and ERK or PI3K inhibition were essentiallyconcordant with those seen in MCF-10A (Fig. 6Aand figs. S6 and S7); differences in the responseof 184A1 cells could be ascribed to efficiency ofpathway inhibition (fig. S7). Similar responseswere also found in an immortalized prostate epi-thelial line, PWR-1E (Fig. 6B and fig. S6). Inagreement with the mammary epithelial lines,PI3K-mTOR inhibition in PWR-1E caused amarkedreduction in p57 abundance, whereas inhibition ofthe ERK pathway strongly increased p57 abun-dance. Nonepithelial human umbilical vein endo-thelial cells (HUVECs) had little basal p57, butnonetheless modestly down-regulated p57 uponPI3K-mTOR inhibition (fig. S8). Thus, stimula-tion of p57 by the PI3K pathway, and inhibitionby the ERK pathway, appears to be a commonprogram in nontumor epithelial cells.

Among breast cancer cell lines examined, nu-clear p57 was difficult to detect above backgroundfluorescence, in agreement with previous observa-tions of low p57 abundance in these lines (31, 32).

A BNo GF IGF-1EGFIGF-1

Fig. 3. Enhancement of IGF-1–stimulated prolifera-tion in p57-depleted cells. (A) HCIF analysis of p57knockdown.MCF-10Acells stably expressing shRNAhairpins targetingp57oranontargetingcontrolhairpinwere grown in the presence of IGF-1 (100 ng/ml) andthe absence of EGF for 2 days. (B) HCIF analysis ofproliferative response in p57-depleted cells. Controland p57 shRNA-expressing MCF-10A cells werecultured in the presence of the indicated growth

factors for 2 days. (C) Quantitation of p57 depletion by shRNA. Histograms represent distributionsof p57 intensity for cells cultured as in (A), and knockdown is shown as the percent change inmedianp57 intensity. (D) Quantitation of pRb-positive p57-shRNA cells cultured in the absence of growthfactors or the presence of IGF-1 (100 ng/ml) or EGF (20 ng/ml) for 2 days. Bars represent the averageof triplicatewells fromone representativeexperiment±SD. *P<0.05; **P<0.01; ***P<0.001; n.s., notsignificant. (E) Growth curve analysis of p57-depleted cells. MCF-10A cells stably expressing theindicated shRNAs were cultured in the presence of the indicated growth factors. Cell counts weredetermined by high-throughput imaging of DAPI-stained cells and normalized to the number of cellsat the time of treatment. Error bars indicate SD of triplicate wells, and results shown are representativeof three independent experiments. (F) Quantitation of pRb-positive p21 +/− or −/− MCF-10A cellscultured as in (D). Bars represent the average of three independent wells ± SD. All data shown arefrom individual experiments representative of at least three independent replicates.

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In MD-MBA-468 and T47D cells, treatment with inhibitors of MEK or ofDNA methylases had limited or no effect on the abundance of p57,whereas the histone deacetylase inhibitor trichostatin A (TSA) induced atwo- to fourfold increase in p57 abundance (Fig. 6, C and D). In the con-text of TSA treatment, p57 abundance was not further increased by MEKinhibition, but was markedly reduced by PI3K-mTOR inhibition. Theseresults suggest that in these breast cancer cells, p57 is primarily suppressedby chromatin modification and requires the PI3K-mTOR pathway for fullinduction. In contrast, in U2OS osteosarcoma cells, which are derivedfrom a nonepithelial lineage, inhibition of either ERK or PI3K-mTORincreased p57 abundance by two- to threefold (fig. S9). Together, theseobservations indicate that the p57 network originally identified in non-tumor cells remains partially intact in tumor cells; in some tumors, p57is suppressed directly by high ERK activity, whereas in others this networkis overridden by chromatin modification.

The frequent loss of p57 in tumors and its ability to specifically limitIGF-1–driven proliferation suggest that it may suppress oncogenicsignaling through the PI3K pathway. To test this hypothesis, we usedMCF-10A cells in which PIK3CA mutations associated with human tu-mors (E545K or H1047R) have been stably integrated at the genomicPIK3CA locus (33). As previously reported, cells harboring either muta-tion maintain low to moderate proliferative activity in the absence of EGF.Notably, the percentage of pRb-positive PI3K mutant cells was enhancedtwo- to fourfold in the absence of EGF by shRNA-mediated depletion ofp57, relative to mutant cells expressing nontargeting shRNA (Fig. 6, Eto H). In agreement, p57 depletion also resulted in an increase in cellnumber (Fig. 6I).

We next assessed the role of p57 in a 3D in vitro model of cellulartransformation (34). In this model, mammary epithelial cells form multi-cellular spheroids resembling mammary acini in vivo, initially dividingbut eventually reaching a state of proliferative arrest. Microarray analysisrevealed that p57 mRNA abundance increased steadily over the course ofmorphogenesis, reaching an 18-fold increase by day 15 (Fig. 7A). Thisincrease occurred simultaneously with proliferative arrest, as assessed byDNA content analysis. In contrast, p21 and p27 mRNAs showed a muchmore modest increase (~2-fold) over the same period. Cells transducedwith p57-targeting shRNA formed acini ~1.5-fold larger than thoseformed by cells transduced with control shRNA vectors (Fig. 7, B andD). This increase in size was accompanied by a marked increase in thenumber of cells positive for the proliferation marker Ki-67 within theacini at day 10 (Fig. 7C). p57-depleted acini frequently displayed filledlumens, which may result from an increase in proliferation exceedingthe rate of cell death in the inner cells. Thus, p57 acts as a barrier touncontrolled proliferation in 3D as well as monolayer culture.

DISCUSSION

Here, we demonstrate a mechanism by which ERK and Akt signals areintegrated in the control of cellular proliferation through differential reg-ulation of the CDK inhibitor p57. Under conditions in which Akt is acti-vated in the absence of detectable ERK activity (for example, in MCF-10Acells treated with insulin or IGF-1 alone or induced to express activatedAkt), p57 abundance increases, suppressing cell proliferation. In contrast,under conditions in which ERK is activated simultaneously with Akt (forinstance, in EGF- or IGF-1–treated cells expressing activated H-Ras), thisincrease in p57 abundance is suppressed, permitting proliferation. Addi-tionally, knockdown of p57 in the context of insulin or IGF-1 treatment orexpression of a stably integrated oncogenic mutant variant of PI3Kenhanced proliferation of mammary epithelial cells. These results indicatethat p57 plays a critical role in suppressing proliferation in mammary ep-

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ithelial cells and that the abundance of p57 is sensitive to the strength ofPI3K-Akt and ERK signaling.

The regulation of p57 by opposing ERK and Akt signals has not pre-viously been described. Previous work has identified numerous integrationpoints downstream of the ERK and Akt pathways, including cyclin D1,c-Myc, and mTOR; however, in these pathways, the integrators respondpositively to both ERK and Akt signals (4, 35–38). In contrast, we de-scribe a mechanism whereby one growth factor–stimulated pathway(ERK) is required to negate the antiproliferative function of another(Akt). Such a regulatory mechanism could serve to “tune” the proliferativeresponse of a cell to the specific intensity of signaling or stimuli. For exam-ple, the proliferative response to activation of the insulin or IGF-1 receptorsis highly context-dependent. Treatment with either of these factors alonedoes not support proliferation in many cell contexts (including in MCF-10A cells grown in serum-free medium) (39–41), whereas either one aloneis sufficient to induce proliferation in other contexts (42, 43). Our data

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Fig. 4. Stimulation of p57 by the Akt network. (A) HCIF images of MCF-10Acells cultured in the presence of IGF-1 (100 ng/ml) and 1 mM Torin-1, 0.5 mMBEZ-235, 0.2 mM GDC-0941, or 20 nM rapamycin for 24 hours. (B) Quan-titation of nuclear p57 by HCIF under the conditions shown in (A). Left:histograms of p57 abundance. Right: frequency of the percentage ofp57-positive (% p57+) cells. The threshold for p57-positive cells wasdefined as in Fig. 2; bars indicate the mean, and error bars the range, ofduplicate measurements. (C) HCIF quantitation of p57 stimulation byinducible myristoylated Akt. MCF-10A cells stably expressing an inducibleAkt variant were cultured in the absence of growth factors for >48 hoursand then stimulated with vehicle (ethanol) or inducer (1 mM 4OHT) in thepresence or absence of EGF (20 ng/ml) for the indicated times. (D) Timingof p57 induction by Akt measured by HCIF. Cells were treated as in (C) forvarying periods of induction before fixation. Curves indicate the average,and error bars SD, of four replicate measurements of median p57 intensity.All data shown are from individual experiments representative of at leastthree independent replicates.

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indicate that changes in p57 abundance resulting from the differential in-duction of Akt and ERK determine, in part, the response to stimulation bythese growth factors. These observations can also be seen as a point of co-operation or synergy between the ERK and the Akt pathways: The full Akt-

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mediated effect on proliferation is only achieved when p57 is concomitant-ly suppressed by ERK. In some contexts, however, control of p57 by ERKand Akt may be superseded by other means of regulation, such as chroma-tin remodeling or genomic imprinting at the CDKN1C locus (44–46).

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d 4A and in (A), pERK, pAkt, and p57 were changes in p57 mRNA abundance. (H) MCF-10A cells cultured in the pres-

Fig. 5. Opposing regu-lation of p57 mRNA bythe Akt and ERK path-ways. (A) HCIF images ofp57 in MCF-10A cells cul-tured with EGF (20 ng/ml)or IGF-1 (100 ng/ml) inthe presence of vehicle(DMSO) or 1 mMMEK in-hibitor (PD0325901) for24 hours. (B) Quantita-tion of p57 intensity forthe conditions shown in(A). Histograms repre-sent distributions of p57abundance, and verticaldashed lines indicatethresholds for p57-positivecells determined as inFig. 2. Percentage val-ues indicate the frequen-cy of p57-positive cells forDMSO- (gray) or inhibitor-treated (blue) conditions.(C) HCIF analysis of p57suppression by activatedH-Ras. MCF-10A cellsstably expressing vectorcontrol or H-RasV12 werecultured in the presenceof IGF-1 or 1 mMMEK in-hibitor (PD0325901) for24 hours. (D) HCIF analy-sis of combined MEKandPI3K-mTORinhibition.MCF-10A cells were cul-tured in the presence ofEGF (20 ng/ml) with orwithout 1 mM PD0325901plus 1 mM Torin-1, or0.5 mM BEZ-235 as indi-cated. (E) Summary ofp57 intensity as a func-tion of pERK and pAktabundance. For all condi-

tions shown in Figs. 2A anmeasured by HCIF and shown as a scatter plot; p57 abundance is repre-sented by the size and intensity of the symbols. (F) Quantitation of p57mRNA abundance by qPCR. Cells were treated with the indicated con-ditions for 24 hours. Values shown are the average of four independentexperiments ± SEM. (G) Quantitation by qPCR of p57 mRNA abundancein response to Akt induction. MCF-10A cells stably expressing inducibleAkt were cultured as in Fig. 4. Values are the average of three independentexperiments ± SEM. (H and I) Time dependence of EGF- and IGF-mediated

ence of IGF-1 were shifted to medium containing IGF-1 and EGF at time0 (red) or maintained in IGF-1 alone (gray). (I) MCF-10A cells cultured inthe presence of EGF and IGF-1 were shifted to medium containing onlyIGF-1 at time 0 (red) or maintained in EGF and IGF-1 (gray). p57 mRNAwas quantitated by qPCR at the indicated times. Values and error barsrepresent the means and SEM of three independent experiments. Unlessotherwise noted, all data shown are from individual experiments repre-sentative of at least three independent replicates.

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The other members of the CIP/KIP family, p21 and p27, display dif-ferent response profiles to ERK, Akt, and other signals (13, 16, 17). Thus,the proliferative response to specific upstream signaling pathways may beregulated by modulating the particular complement of CIP/KIP familymembers present in the cell. Our examination of different cell lineagessuggests that p57 may be of particular importance in epithelial cells.The importance of maintaining such a balance in physiological situationsis supported by our finding that down-regulation of p57 causes hyperpro-liferative effects in 3D acini, a model for growth regulation of ductalstructures in mammary tissue.

The ERK-Akt-p57 circuit could also serve as sensor for imbalanced oroncogenic signaling. Many cancers carry mutations in the PIK3CA iso-form of PI3K or loss of PTEN. On the basis of our studies, such altera-tions would be predicted to induce activation of the Akt pathway withoutactivating ERK, leading to induction of p57, and preventing inappropriatecell proliferation. This hypothesis would suggest that the barrier imposed

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by p57 would need to be overcome at some point in the progression oftumors driven by PI3K network hyperactivation. The ability of the ERKpathway to suppress p57 may create a selective pressure favoring concom-itant mutations in the PI3K and ERK pathways (47). A careful bioinfor-matic study of the mutational status of these pathways in conjunction withp57 status will be necessary to evaluate this hypothesis.

Consistent with its tumor suppressor role, p57 is frequently inactivatedthrough multiple mechanisms in cancer (20). Most breast cancers displaydecreased p57 abundance at the histological level (48), and p57 expressionis frequently suppressed by epigenetic silencing of the CDKN1C locus inboth breast and ovarian cancers (31, 49, 50). Analysis of microarray datafrom a large panel of breast cancer cell lines (32) revealed that 46 of 49tumor-derived lines displayed low p57 mRNA abundance relative to thetwo nontumor cell lines included in the panel, MCF-10A and MCF-12A.In the immortalization process of normal mammary epithelial cells, p57 isup-regulated after bypass of replicative senescence but silenced upon full

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Fig. 6. Regulation ofp57 in epithelial andcancer cell lines. (Aand B) Quantitation ofp57 by HCIF in (A)MCF-12A mammaryepithelial cells and(B) PWR-1E prostateepithelial cells treatedwi th the indicatedgrowth factors and in-hibitors of MEK (PD)or PI3K-mTOR (BEZ)for 24 hours. The per-centage of p57-positivecells was determinedas in Fig. 2. Error barsrepresent the SD of trip-licate wells. (C and D)Quantitation of p57 byHCIF in T47D (C) andMD-MBA-468 (D) cellscultured in the presenceof the indicated inhibi-tors of MEK (PD), PI3Kand mTOR (BEZ), his-tone deacetylases [tri-chostatin A (TSA)], orDNAmethyltransferases[5′-aza-deoxycytidine(AZA)] for 24 hours.Bars represent the av-erage p57 intensity oftriplicate wells ± SD.Under some conditions(marked by †), a large

percentage of cells committed apoptosis; p57 fluorescence values werederived from surviving cells. (E) HCIF images of shRNA-mediated p57 de-pletion in PIK3CA-mutant cells. MCF-10A cells containing mutant alleles ofPIK3CA (E545K or H1047R) at the endogenous locus and stably ex-pressing nontargeting (NT) or p57-specific shRNA were cultured in the ab-sence of EGF for 2 days. (F) HCIF images of Rb phosphorylation in PIK3CAmutant cells. Cells were cultured as in (E). (G and H) Quantitation of pRb

positivity for cells grown in the absence of EGF for 2 days (G), or theabsence or presence of EGF (20 ng/ml) for 1 day (H). Bars representthe average of three independent wells ± SD. (I) Growth curve analysis ofp57-depleted PIK3CA mutant cells. Cells were cultured as in (E), and cellcountsweredeterminedbyHCIF analysis ofDAPI-stainednuclei. Errorbarsrepresent the SD of triplicate wells. All data shown are derived from individ-ual experiments representative of at least three independent replicates.

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immortalization (51). Together, these data suggest that the regulation ofp57 by ERK and Akt plays a role in suppressing proliferation in responseto aberrant signaling during the initial stages of carcinogenesis; this reg-ulatory function is then lost in the later stages of transformation when theCDKN1C locus is silenced.

Although our data demonstrate regulation of p57 at the mRNA levelby EGF and IGF-1 through the ERK and Akt pathways, respectively, theintermediate links between these kinases and p57 mRNA regulation re-main to be elucidated. The ERK pathway activates various transcription-al modifiers, including AP-1 (activating protein 1), c-Myc, Elk-1, andEZH-2 (enhancer of zeste homolog 2), whereas the Akt pathway controlsFoxO3a, CREB (cAMP response element–binding protein), AP-1, c-Myc,and others. The histone methyltransferase EZH-2, which has been impli-cated in repression of p57 expression, is activated transcriptionally byERK (52) and inactivated through phosphorylation by Akt (53), makingit a potential mediator of p57 regulation. Another attractive mechanism fordown-regulation of p57 by ERK is the c-Myc–induced miR-221/222,which targets p57 (22, 23). However, because both IGF-1 and EGF increasec-Myc abundance in MCF-10A cells (with EGF doing so more strongly;see Fig. 1), this mechanism is insufficient to explain the difference betweenIGF-1 and EGF in p57 regulation, unless miR-221/222 induction is highlysensitive to different amounts of c-Myc. It is likely that a number of distincttranscriptional and posttranscriptional regulators contribute to p57 regula-tion by the ERK and Akt pathways.

Because insulin and Akt suppress ERK activation (10), it is possiblethat Akt induction of p57 could occur through decreased ERK suppres-sion of p57 mRNA transcription. However, several results argue againstthis mechanism and indicate that p57 induction requires a positive inputfrom the Akt pathway, not simply suppression of ERK signaling. First,myrAkt is able to increase the abundance of p57 mRNA and protein incells deprived of growth factors for 48 hours, where there is no detectablepERK signal. Second, induction of p57 by MEK inhibition in the contextof EGF stimulation is blocked by PI3K-mTOR inhibition.

A major challenge in signal transduction research is to understand howcomplex networks integrate multiple signals to reach a phenotypic cell fatedecision. Computational models have been developed that focus on predict-ing the degree and kinetics of ERK and Akt activation in response to receptoractivation or pharmacological inhibition (54, 55). However, the connectionsdownstream of ERK and Akt that determine cell cycle control and prolifer-ative outcome have yet to be extensively modeled. Developing a quantitativeunderstanding of the connection between upstream signals and downstreamphenotypes will be essential for predicting the efficacy of inhibitors targetedto these pathways, and these models will depend on accurate “wiringdiagrams” of the multiple integration points between ERK and Akt pathwayeffectors. The mechanism described here adds a new integration point criticalfor epithelial cell cycle regulation by these signals; additional studies will beneeded to understand the relative importance of this and other integrationmechanisms, which will likely be highly context-dependent.

MATERIALS AND METHODS

Cell cultureMCF-10A mammary epithelial cells and the clonal derivative 5E (56)were cultured as previously described (34) in MCF-10A growth medium[Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Invitrogen) supple-mented with 5% horse serum, EGF (20 ng/ml), insulin (10 mg/ml), hydro-cortisone (0.5 mg/ml), cholera toxin (100 ng/ml), penicillin (50 U/ml), andstreptomycin (50 mg/ml)]. Experimental treatments with varying concen-trations of growth factors were prepared in GM-GFS medium [DMEM/F12

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Fig. 7. Hyperproliferation in p57-depleted cells in 3D culture. (A) Micro-array and proliferation analysis of CDKN1 family mRNA abundance inMCF-10A cells during 3D morphogenesis. mRNA abundance for p21,p27, and p57 is shown normalized to the amount on day 2 ± SEM oftriplicate measurements (left vertical axis). The percentage of cells with>2N DNA content (percent in G2-S, gray) was assessed by flow cytom-etry after trypsinization and staining with propidium iodide (right verticalaxis). Data are from one experiment representative of two independentreplicates. (B) Phase-contrast images of acinar structures formed byMCF-10A cells transduced with control or p57-targeting shRNA vectorsat day 10 in the 3D morphogenesis assay. Data are from one experimentrepresentative of four independent replicates. (C) Immunofluorescencedetection of Ki-67 in control or p57-shRNA acinar structures at day 10 of3D culture. Data are from one experiment representative of two inde-pendent replicates. (D) Quantitation of acinar structure size for controland p57-shRNA acinar structures at day 17 in 3D culture. Data are fromone experiment representative of three independent replicates.

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supplemented with 0.3% bovine serum albumin, hydrocortisone (0.5 mg/ml),cholera toxin (100 ng/ml), penicillin (50 U/ml), and streptomycin (50 mg/ml)].HUVECs were obtained from Invitrogen and 184A1 cells from the Amer-ican Type Culture Collection. MCF-12A and 184A1 cell lines were culturedin the same growth medium as MCF-10A cells, PWR-1E cells in keratin-ocyte serum-free medium (Invitrogen), HUVECs in M200 + LSGS (low-serum growth supplement) (Invitrogen), U2OS cells in McCoy’s 5Amedium supplemented with 10% fetal bovine serum (FBS), and T47Dand MD-MBA-468 cells in RPMI medium supplemented with 10% FBS.

ReagentsThe following primary antibodies were used: antibodies against p21 [CellSignaling (CS), BD Biosciences (BD)], p27 (CS, BD), p57 [Santa Cruz(SC), CS], pERK1/2 (CS), pRb-S780 (SC), pAkt-S473 (CS), FoxO3a (CS),c-Fos (CS), c-Myc (CS), pS6 (CS), c-Jun (CS), Egr-1 (CS), Fra-1 (SC),Ets-1 (SC), Elk-1 (SC), and Alexa Fluor 488, 555, and 647 (Invitrogen).The following compounds and small-molecule inhibitors were used:IGF-1 (R&D Systems), DMSO (Sigma), PIK-90 (Axon Medchem),GDC-0941 (Axon), BEZ-235 (Axon), rapamycin (Calbiochem), PD0325901(Calbiochem), PD98059 (Calbiochem), Torin-1 (N. Gray), TSA (Calbiochem),and 5′-aza-deoxycytidine (AZA) (Calbiochem).

DNA vectors and generation of cell linesThe pMSCV-puro–ER–myrAkt retroviral vector was generated frompWZLmyrAkt-HA-ER (a gift from R. Roth, Stanford University, Stanford,CA). pBabe-puro and pBabe-puro–HRasV12 vectors were previously de-scribed (57). Lentiviral vectors (pLKO.1puro) encoding anti-p57 shRNAsA, B, C, and D were obtained from the RNAi Consortium. To generatecell lines, we infected MCF-10A or MCF-10A-5E cells with the retroviralor lentiviral vectors and selected stable populations with puromycin (2 mg/ml).

High-content immunofluorescence microscopyCells cultured in 96-well optical-bottom plates (Corning) were fixed with2% paraformaldehyde and permeabilized with methanol at −20°C. OdysseyBlocking Buffer (LI-COR) was used for blocking and antibody dilution.After incubation with primary and secondary antibodies, plates were imagedon a CellWoRx Scanner (Applied Precision). Three to four image fieldswere collected in each well of a 96-well plate (uniform count per experi-ment) and processed in MATLAB (MathWorks) with segmentation rou-tines derived from CellProfiler (58), with Otsu thresholding for nuclearidentification and an annulus of five pixels radial to the nucleus for thecytoplasmic region. Routine inspection of cell segmentation typicallyrevealed successful identification of nuclei for greater than 85 to 90%of cells. After background estimation and subtraction based on an im-age opening algorithm, single-cell fluorescence intensity values foreach channel were calculated as the mean pixel value for the nuclearor cytoplasmic regions.

Growth curve analysisCells were seeded in optical-bottom 96-well plates and cultured underthe indicated conditions, and medium was replaced every 2 days. At theindicated time points, cells were fixed in 100% methanol, stained with4′,6-diamidino-2-phenylindole (DAPI), and imaged with >95% well cov-erage. Nuclei were counted computationally after segmentation withImageRail (59). Cell counts were normalized to the number of cells onduplicate plates fixed at the time of initial treatment (day 0).

Quantitative real-time PCRTotal RNA was prepared from cells with TRIzol Reagent (Invitrogen).Complementary DNA (cDNA) was synthesized from 2 mg of RNA with

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the SuperScript First-Strand Synthesis System (Invitrogen). Real-timePCR was performed with an ABI Prism 7900HT Fast Real-Time PCRSystem with gene-specific primers (CDKN1C: forward 5′-GCGGCGAT-CAAGAAGCTG-3′, reverse 5′-CGACGACTTCTCAGGCGC-3′;RPLP0: forward 5′-ACGGGTACAAACGAGTCCTG-3′, reverse 5′-CGACTCTTCCTTGGCTTCAA-3′) and Power SYBR Green PCRMaster Mix (Applied Biosystems). Relative mRNA abundances weredetermined and normalized to the ribosomal protein RPLP0.

3D morphogenesis assays3D culture of MCF-10A cells in Matrigel (BD Biosciences) was per-formed as previously described (34). MCF-10A control and p57knockdown acini were cultured in assay medium [MEM/F12 supplementedwith 2% horse serum, EGF (5 ng/ml), insulin (10 mg/ml), hydrocortisone(0.5 mg/ml), cholera toxin (100 ng/ml), penicillin (50 U/ml), streptomycin(50 mg/ml), and 2% Matrigel] and refed every 4 days.

Microarray and immunofluorescence analysisMicroarray and immunofluorescence analysis of 3D acinar structures wereperformed as described previously (34, 60). Indirect immunofluorescenceand phase imaging were performed on a Nikon TE300 microscopeequipped with a mercury lamp and a charge-coupled device camera.

Statistical analysisFor qPCR experiments, averaged values represent the averages fromindependent replicate experiments and error bars the SEM. For HCIFexperiments, values from individual wells containing 2000 to 10,000 cellswere represented as the median single-cell intensity value. Because of day-to-day variations in fluorescence values, quantitation of HCIF experimentsis shown as the averaged median fluorescence value of triplicate samplescollected on the same day, and error bars represent the SD. Allexperiments were repeated independently at least three times to confirmthat the data shown were representative in both magnitude and overalltrend. P values were calculated by Student’s two-tailed t test and indicatedas follows: *P < 0.05; **P < 0.01; ***P < 0.001.

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/5/214/ra19/DC1Fig. S1. HCIF images of p21 under various growth factor conditions.Fig. S2. Covariate single-cell analysis of p57/p21 and pRb.Fig. S3. Immunoblot analysis of p57 depletion.Fig. S4. Proliferative and signaling response to Akt induction.Fig. S5. Immunoblot analysis of changes in p57 protein abundance.Fig. S6. HCIF images of p57 in MCF-12A and PWR-1E epithelial cell lines.Fig. S7. HCIF analysis of p57 regulation in 184A1 mammary epithelial cells.Fig. S8. HCIF analysis of p57 in HUVECS.Fig. S9. HCIF analysis of p57 regulation in U2OS osteosarcoma cells.

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Acknowledgments: We thank the Nikon Imaging Center at Harvard Medical School andthe Harvard Institute for Chemical and Cell Biology for help with light microscopy. The 5Eclonal derivative of MCF-10A cells was provided by K. Janes; p21−/−, p21+/−, and thePIK3CA mutant knock-in MCF-10A cell lines by B. H. Park; and the inducible Akt constructby R. Roth. We thank T. Michel and M. Niepel for helpful discussions. Funding: This workwas supported by the U.S. NIH (5-R01-CA105134-07 to J.S.B.) and by a Department ofDefense Breast Cancer Research Program postdoctoral fellowship (W81XWH-08-1-0609to J.G.A.). E.S.L. contributed to this manuscript as an employee of Millennium Pharma-ceuticals Inc. Author contributions: D.T.W., T.S., N.L.S., E.S.L., and J.G.A. performedthe experiments and analyzed the data; B.M. and J.G.A. performed computational anal-ysis; D.T.W., T.S., G.B.M., J.S.B., and J.G.A. designed and interpreted the experiments;and D.T.W., G.B.M., J.S.B., and J.G.A. wrote the paper. Competing interests: Theauthors declare that they have no competing financial interests.

Submitted 4 March 2011Accepted 10 February 2012Final Publication 6 March 201210.1126/scisignal.2001986Citation: D. T. Worster, T. Schmelzle, N. L. Solimini, E. S. Lightcap, B. Millard, G. B. Mills,J. S. Brugge, J. G. Albeck, Akt and ERK control the proliferative response of mammaryepithelial cells to the growth factors IGF-1 and EGF through the cell cycle inhibitor p57Kip2.Sci. Signal. 5, ra19 (2012).

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