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Tumor Biology and Immunology

AMPK–Akt Double-Negative Feedback Loop inBreast Cancer Cells Regulates Their Adaptationto Matrix DeprivationManipa Saha1, Saurav Kumar1, Shoiab Bukhari1, Sai A. Balaji1, Prashant Kumar2,Sravanth K. Hindupur1, and Annapoorni Rangarajan1

Abstract

Cell detachment from the extracellular matrix triggers anoi-kis. Disseminated tumor cells must adapt to survive matrixdeprivation, while still retaining the ability to attach at sec-ondary sites and reinitiate cell division. In this study, weelucidate mechanisms that enable reversible matrix attachmentby breast cancer cells. Matrix deprival triggered AMPK activityand concomitantly inhibited AKT activity by upregulating theAkt phosphatase PHLPP2. The resultant pAMPKhigh/pAktlow

state was critical for cell survival in suspension, as PHLPP2silencing also increased anoikis while impairing autophagyand metastasis. In contrast, matrix reattachment led to Akt-mediated AMPK inactivation via PP2C-a-mediated restoration

of the pAkthigh/pAMPKlow state. Clinical specimens of primaryand metastatic breast cancer displayed an Akt-associated geneexpression signature, whereas circulating breast tumor cellsdisplayed an elevated AMPK-dependent gene expression signa-ture. Our work establishes a double-negative feedback loopbetween Akt and AMPK to control the switch between matrix-attached and matrix-detached states needed to coordinate cellgrowth and survival during metastasis.

Significance: These findings reveal a molecular switch thatregulates cancer cell survival during metastatic dissemination,with the potential to identify targets to prevent metastasis inbreast cancer. Cancer Res; 78(6); 1497–510. �2018 AACR.

IntroductionMetastasis accounts for the vast majority of cancer-associated

deaths. The metastatic process involves detachment of cells fromthe primary site of tumor initiation, entry into the blood streamorthe lymphatics, exit from the circulation and reattachment atdistant sites to spawn metastatic growth (1). Integrins mediatecell adhesion to the extracellular matrix that provides growth andsurvival signals (2), whereas matrix deprivation leads to pro-grammed cell death termed "anoikis" (3). Therefore, detachedtumor cellsmust develop resistance to anoikis, while retaining theability to reattach and grow at a distal site to spawn a successfulmetastasis. Yet, little is known about cellular signaling pathwaysthat coordinate cell growth and stress-survival signals during theattachment–detachment cascade of metastatic colonization.

The serine/threonine protein kinase Akt (also known as PKB)regulates several cellular processes, including proliferation, sur-

vival, and metabolism, and plays a major role in tumor progres-sion (4). Akt is recruited to the plasma membrane by binding toPIP3 and is subsequently phosphorylated by PDK1 and mTORcomplex 2 (mTORC2) at T308 and S473, respectively, leading toits full activation. Conversely, Akt signaling is attenuated bydephosphorylation of these sites by protein phosphatase 2A(PP2A) and pleckstrin homology domain leucine-rich repeatprotein phosphatases (PHLPP 1 and 2; ref. 5). Upon activationby growth factor signaling, Akt promotes anabolic processesincluding lipid biosynthesis and protein translation, thus drivingcell growth and proliferation.

In contrast, the AMP-activated protein kinase (AMPK) is acti-vated under metabolically stressed conditions and brings aboutcellular homeostasis by switching on energy-generating catabolicprocesses like fatty acid oxidation and glycolysis, while inhibitingenergy-consuming anabolic pathways including carbohydrate,lipid, and protein biosynthesis (6–8). AMPK is a heterotrimericprotein consisting of a, b, and g subunits (encoded by a1, a2; b1,b2; and g1, g2, g3). It is allosterically activated by AMP andpositively regulated by phosphorylation of T172 residue byupstream kinases LKB1 and CaMKKb, while negatively regulatedby dephosphorylation (9, 10). Although considered a tumorsuppressor owing to its growth retarding effects, recent studieshave identified context specific protumorigenic roles for AMPK bypromoting cell survival under glucose deprivation and hypoxiastress (11, 12).

Under matrix-deprivation stress, Akt activation is sufficient foranoikis resistance in immortalized MDCK cells (13). ErbB2-over-expressing breast cancer cells show increased dependence on Aktfor anchorage-independent growth (14). In contrast, pharmaco-logic inhibition of the PI3K/Akt pathway failed to render T-47Dbreast cancer cells sensitive to anoikis (15). Thus, the role of Akt in

1Department of Molecular Reproduction, Development and Genetics, IndianInstitute of Science, Bangalore, India. 2Institute of Bioinformatics, InternationalTechnology Park, Whitefield, Bangalore, India.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M. Saha and S. Kumar contributed equally to this article.

Current address for Sravanth K. Hindupur: Biozentrum, University of Basel,Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.

CorrespondingAuthor:Annapoorni Rangarajan, Indian Institute of Science, LabGA 10 MRDG IISc Bangalore, Bangalore 560012, Karnataka, India. Phone: 91-80-22933263; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-2090

�2018 American Association for Cancer Research.

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anoikis resistance remains to be fully understood. On the otherhand, recent work from our laboratory and that of others hasshown matrix deprivation-triggered activation of AMPK and itscritical role in anoikis resistance in breast cancer cells (16–18).Thus, independent studies have implicated Akt and AMPK inanoikis resistance, although they have opposing effects on cellulargrowth and metabolism.

Synergistic and antagonistic relationship between Akt andAMPK has been documented under different cellular contexts;however, little is known about their interplay in maintaining theadherent versus detached states of cells. Intriguingly, we showhere that detachment-triggered AMPK concomitantly repressesAkt activity. We identify a novel AMPK-mediated PHLPP2 upre-gulation that inactivates Akt to promote AMPK-induced autop-hagy and that inhibits anoikis in suspension. Finally,we show thatmatrix reattachment triggers Akt activity, which in turn repressesAMPK through PP2C-a. Our data, thus, identify a novel, recip-rocal, inhibitory relationship between AMPK and Akt that reg-ulates adaptation to matrix detachment.

Materials and MethodsPrimary cells and culture conditions

Primary breast tissues (cancer and adjacent normal) obtainedfrom the Kidwai Memorial Institute of Oncology (KMIO), Ban-galore, as per IRB and in compliance with ethical guidelines ofKMIO and the Indian Institute of Science (IISc), were processedinto single cells and cultured as described previously (16, 19) inserum-free media containing 10 ng/mL hEGF, 1 mg/mL hydro-cortisone, 10 mg/mL insulin, 4 ng/mL heparin and B27. Singlecells were seeded in regular TC plates for adherent culture or inultralow attachment plates (Corning Inc.) for mammosphereculture (16).

Cell lines and cell culture conditionsBreast cancer cell lines MDA-MB-231, MCF7, BT474 (from

ATCC in 2016, and validated by STR analysis); A549 and H460(lung), LN229 (glioma), Hep3B (liver), and HeLa (cervical)cancer cell lines (obtained as kind gifts) were cultured in DMEM(Sigma-Aldrich) supplemented with 10% FBS containing peni-cillin and streptomycin, at 37�C and 5%CO2. Cell lines were usedfor experiments within 8 passages after thawing. For short-termsuspension cultures of 8 hours and 10 minutes, cancer cells wereseeded on dishes coated with 1 mg/mL poly-2-hydroxyethyl-methacrylate (Poly-HEMA; Sigma-Aldrich) dissolved in absoluteethanol. For 48 hours of suspension culture, cells were seeded ondishes coated with 1% noble agar (Sigma-Aldrich). Long-termanchorage-independent (AI) colony formation assay was under-taken by admixing 1� 105 cancer cells either with a slurry of 1.5%methyl cellulose or in 0.3% soft agar, and layered over 0.6%nobleagar. AI colonies frommethyl cellulose were harvested for immu-noblotting after 7 days or counted after 15 days from 15 randomfields of 10� magnification in each 35-mm dish.

Plasmids, transfection, and generation of stable cell linesGFP-HA-Akt-T308D S473D (#39536; originally submitted by

Dr. Julian Downward), referred to as GFP Akt DD in figures, andmyc AMPKa2 K45R (#15992; originally submitted by Dr. MorrisBirnbaum), referred to as dominant-negative (DN) AMPK, wereprocured from Addgene. HA myr-Akt and GFP CA CaMKK wereprovided by Dr. Joseph Testa and Dr. Grahame D. Hardie,

respectively, as kind gifts. shRNAs against PHLPP2 (RHS4531-EG23035) and the corresponding control nontargeting shRNA inpGIPZ vector (NT); and inducible shRNA against AMPKa2(V2THS_57674) and the corresponding control empty pTRIPZvector (EV) were procured from Dharmacon. Lipofectamine(Invitrogen) was used to transfect plasmid DNA into cells.

MDA-MB-231 cells stably expressing GFP-HA-Akt-T308DS473D were generated by transfection followed by FACS-basedsorting for GFP-expressing cells; cells stably expressing HA myr-Akt were generated by cotransfecting a puromycin resistanceplasmid at a 10:1 ratio followed by selection with puromycin(0.5 mg/mL) treatment. MDA-MB-231 cells stably expressingspecific shRNAs were generated by selection with puromycinfollowed by sorting cells for high GFP (in case of plasmids inpGIPZ vector) or high RFP (in case of plasmids in pTRIPZ vector)expression.

siRNA oligos against Akt (targeting both isoforms Akt1 andAkt2 [6211 and 6510]) were purchased from Cell SignalingTechnology and transfected using oligofectamine (Invitrogen).

Pharmacologic compoundsPharmacologic compounds used in cell culture include the

AMPK inhibitor 6-[4-(2-piperidin-1-ylethoxy-phenyl)]-3-pyridin-4-yl-pyrrazolo [1, 5-a]-pyrimidine (compoundC;Cat.No. 171260;10 mmol/L; referred to as CC in figures), PI3K/Akt inhibitorLY294002 (Cat. No. 440202; 20 mmol/L; referred to as LY infigures), Akt inhibitor Akti VIII (Cat. No. 124018; 10 mmol/L), andMG132 (Cat. No. 474790; 10mmol/L) from Calbiochem (Merck),AMPK activator A-769662 (100 mmol/L; referred to as A76 infigures) from the University of Dundee, Scotland, cycloheximide(Cat. No. C7698; 0.1 mg/mL; referred to as CHX) and lysosomalinhibitor chloroquine (Cat. No. C6628; 50 mmol/L; referred to asCQ in figures) from Sigma-Aldrich. Dimethyl sulfoxide (DMSO)was used as vehicle control for all compounds except cyclohexi-mide, which was dissolved in water. In experiments carried out insuspension, adherent cells were pretreated with the respectivechemicals for 2 hours prior to being subjected to suspension inthe continued presence of the chemicals.

Immunoblotting and immunoprecipitationFor immunoblotting, whole-cell lysates were prepared using

lysis buffer containing 50 mmol/L Tris, 50 mmol/L sodiumfluoride, 5 mmol/L sodium pyrophosphate, 1 mmol/L EDTA,1 mmol/L EGTA, 1% Triton X-100, 0.2 mg/mL DTT, 0.2 mg/mLbenzamidine, and protease inhibitor (Roche) on ice. Proteinconcentrationwas estimated using Bradfordmethod, equal quan-tity of protein (30–50 mg) per lane was resolved by SDS-PAGEafter boiling with sample buffer for 3 minutes at 100�C. Proteinswere transferred to PVDFmembrane andprobedwith appropriateantibodies. The membrane was incubated overnight withprimary antibody at 4�C followed by washes in TBST, and incu-bated for 2 hours with HRP-conjugated secondary antibody atroom temperature. Chemiluminescence (using ECL substratefrom Thermo Fisher Scientific) was used to visualize proteinbands. The membranes were stripped using 1 mol/L Trisbuffer (pH 6.8) containing 2% sodium dodecyl sulfate and0.7% b-mercaptoethanol and then used for repeated probingwith subsequent antibodies, following washes and blocking.

Multipanel blots were assembled by reprobing the sameblot for successive antibodies or by running the same lysatemultiple times; a-tubulin served as loading control for each run.

Saha et al.

Cancer Res; 78(6) March 15, 2018 Cancer Research1498




Representative immunoblots showdata consistentwithminimallythree independent experiments. Densitometric analyses of Westernblots were performed using the Multigauge V2.3 software. Rela-tive protein levels were quantified by normalizing to loadingcontrol. Primary antibodies used in the study were againstpAMPKaT172, pACCS79, pAktS473, pAktT308, pPRAS40T246, totalAMPKa (that recognizes both AMPKa 1 and 2 isoforms),AMPKa2, ACC, Akt, PRAS40, PP2C-a, myc tag, HA tag, cleavedcaspase-3, LC3B, GFP, Ubiquitin (Ub), IgG (Cell Signaling Tech-nology), a Tubulin (Calbiochem), PHLPP2 (Abcam), PP2A-Aa/b, and PPM1E (Santa Cruz Biotechnology). HRP-conjugatedanti-mouse and anti-rabbit antibodies were obtained from Jack-son ImmunoResearch Laboratories. For immunoprecipitationexperiments, cells were lysed in buffer containing 20 mmol/LTris (pH 8), 137mmol/L NaCl, 10% glycerol, and 1%Nonidet P-40, supplemented with protease inhibitors, sodium fluoride, andsodium orthovanadate. Cellular protein (1 mg) was incubatedwith one of control IgG, anti-PP2A-Aa/b, anti-PPM1E, or anti-PP2C-a antibody and 15 mL of protein-A sepharose beads for 12hours at 4�C on end-on rocker. The immune complexes wereprecipitated by centrifugation at 1300 rpm for 5 minutes at 4�C.The precipitates were washed with Nonidet P-40 lysis buffer sup-plemented with 1 mol/L NaCl. Immune complexes were resus-pended in50mLof samplebuffer andanalyzedby immunoblotting.

Caspase-3 activity assayCaspase-3 activity was measured by using a CaspGLOW Red

Active caspase-3 activity kit from Bio Vision (K-193) as per themanufacturer's instructions. Briefly, 1 � 106 cells were stainedwith 1mL of Red-DEVD-FMK for 30minutes at 37�C and 5%CO2.Cells were washed thrice using wash buffer and analysis was doneusing BD FACS-CantoII (Becton & Dickinson) equipped with a488-nm Coherent Sapphire Solid State laser. Red (564–606 nm)fluorescence emission from 104 cells was measured after illumi-nation with blue (488 nm) excitation light. Data were analyzedusing Summit software V5.2.1.12465.

Acridine orange assay for autophagyAutophagy is characterized by formation of acidic vesicular

organelles (AVO). To detect AVOs, acridine orange (AO) wasused. Acidic compartment causes accumulation of AO, whichgives bright red fluorescence upon excitation by 488-nm laser.Measurement of red fluorescence is proportional to increase inAVOs (20).

Cells (2 � 105) were subjected to suspension culture for 48hours, after which they were trypsinized, counted, and 1 � 105

cells were stained with acridine orange (1 mg/mL) for 15 minutesat 37�C in DMEM þ 10% FBS. Post staining, cells were washedthrice in PBS. The cells were analyzed in BD FACSCantoII (Becton& Dickinson) equipped with a blue (488 nm) Coherent SapphireSolid State laser. Red (564–606 nm) fluorescence emission from104 cells was measured. Data were analyzed using SummitV5.2.1.12465 software.

Phosphatase activity assayFor phosphatase activity assays, cell lysate was prepared in

phosphatase activity buffer (20 mmol/L imidazole–HCl, pH7.0, 2 mmol/L EDTA, 2 mmol/L EGTA, and protease inhibitorcocktail). PP2A-Aa/b, PPM1E, and PP2C-a activities were mea-sured using Ser/Thr phosphatase assay kit (Cat. No. 17-127;Millipore) according to the manufacturer's protocol. Briefly,

500 mg of total protein lysate prepared from cells under attached(Att), suspension (Sus), and reattached (Re-Att) conditions wasindependently immunoprecipitated with antibodies againstPP2A-Aa/b, PPM1E, or PP2C-a and the activity of the phospha-tases was estimated colorimetrically by measuring the releasedphosphate from threonine phosphopeptide (K-R-pT-I-R-R) withMalachite Green Phosphate detection solution (absorbance mea-sured at 640 nm).

Microarray and data analysisMDA-MB-231 cells cultured in attached or suspension condi-

tions for 24 hours, and MDA-MB-231 cells stably expressingshAMPKa2, shPHLPP2, and GFP Akt DD cultured in suspensionwere harvested for microarray experiments. Total cellular RNA wasisolated using an RNeasy minikit (Cat No.74104; Qiagen) accord-ing to the manufacturer's protocol. The RNA samples were labeledwithCy3usinganAgilent'sQuick-AmplabelingKit (Cat.No. 5190-0442) and subjected to hybridization on Agilent's In situ Hybrid-ization Kit (Cat No.5188-5242). For analysis, a list of signaturegenes was taken fromAmiGOGeneOntology Consortium (http://amigo.geneontology.org/amigo), Profiler PCR Array list Qiagen,and further validated by KEGG (http://www.genome.jp/kegg/pathway.html) or PubMed (https://www.ncbi.nlm.nih.gov/pubmed/).Heat map of individual and combined data sets was generatedusing an online tool Morpheus (https://software.broadinstitute.org/morpheus/#). Unsupervised clustering was performed toobtain function based gene expression signature. Genes of interestare color coded; "red" in theheatmaprepresentsupregulatedgenes,whereas "green" represents downregulated genes.

Microarray raw data for primary breast tumor, circulatingtumor cells (CTC), and metastases were taken from Gene Expres-sion Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/), withGEO IDs GSE43837, GSE99394, and GSE56493. These raw datafiles were processed and expression values were calculated in log2scale. Further, the data were normalized using Z-score.Microarrayfor GSE43837 set was performed on GPL1352 [U133_X3P]Affymetrix Human X3P Array platform, GSE99394 set was per-formedon [HTA-2_0]AffymetrixHumanTranscriptomeArray 2.0[transcript (gene) version], and GSE56493 set was performed on[U133_X3P] Affymetrix Human X3P ArrayRosetta/Merck HumanRSTA Custom Affymetrix 2.0 microarray [HuRSTA-2a520709]platform. Official gene symbols for the corresponding probeswere retrieved using DAVID (https://david.ncifcrf.gov/summary.jsp) and expression array platform. Heat maps of combineddatasets were generated using Morpheus (https://software.broadinstitute.org/morpheus/#). Semisupervised clustering was per-formed to obtain function-based gene expression signatures. Boxplots were plotted using GraphPad Prism 5.0 software.

Metastasis assay in miceAll animal experiments were reviewed and approved by the

Institutional Animal Ethics committee of IISc, Bangalore. Cells(2� 106) were resuspended in 50 mLDMEM and injected into thelateral tail vein of 5-week-old female NOD-SCIDmice. Mice weresacrificed after 2 months of injection, and lungs and liver weredissected to check for metastatic nodules.

Statistical analysisStatistical analyses were performed using GraphPad Prism 5.0

software using Student t test. All data are presented as mean �SEM, where �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

Reciprocal Interaction between AMPK and Akt

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http://amigo.geneontology.org/amigo

http://amigo.geneontology.org/amigo

http://www.genome.jp/kegg/pathway.html

http://www.genome.jp/kegg/pathway.html

https://www.ncbi.nlm.nih.gov/pubmed/

https://software.broadinstitute.org/morpheus/#


https://www.ncbi.nlm.nih.gov/geo/

https://david.ncifcrf.gov/summary.jsp

https://david.ncifcrf.gov/summary.jsp




ResultsMatrix deprivation leads to Akt inactivation concomitant toAMPK activation

To begin to understand the interplay between Akt and AMPKduring the attachment–detachment cascade ofmetastasis, we firstinvestigated their relative activities under these conditions inbreast cells. For this, we measured phosphorylation of Akt atS473 (pAktS473) and phosphorylation of AMPKa at T172(pAMPKaT172) as surrogate measures of their activities (5, 21),respectively. We recently reported AMPK activation in mammo-spheres formed by normal HMECs and spheroids formed bybreast cancer cells (16). Therefore, we first investigated the statusof Akt activity in these 3-dimensional spheroids. Interestingly,concomitant with increase in the levels of pAMPKaT172, our studyrevealed a significant reduction in the levels of pAktS473 inanchorage-independent spheroids generated by HMECs (Fig.1A), breast cancer cell lines MDA-MB-231 and MCF7 (Figs. 1Band C), and primary breast cancer-derived cells (SupplementaryFig. S1A), compared with their respective adherently growing

cultures. The levels of total Akt and total AMPKa proteinsremained unchanged between these two conditions in all thecell types.

Consistent with mammospheres, subjecting MDA-MB-231cells tomatrix detachment for 8 hours also resulted in a significantdecrease in pAktS473 levels concomitant with increase inpAMPKaT172 levels (Fig. 1D). Further, phosphorylation of Aktat T308, which contributes to full Akt activation, was also reducedin suspension (Fig. 1E). In keeping with their active phosphor-ylation status, we also observed a decrease in the phosphorylationof PRAS40, an Akt substrate (22), and an increase in the phos-phorylation of ACC, an AMPK substrate (Fig. 1E; SupplementaryFig. S1B; ref. 23). We obtained similar results as early as 10minutes of suspension culture (Supplementary Fig. S1B). In vitrokinase assays further substantiated the reduction in Akt activityand increase in AMPK activity in suspension (Supplementary Fig.S1C). We also observed elevated pAMPKaT172 and reducedpAktS473 levels in several other matrix-deprived cancer cell linesof different epithelial origin (Supplementary Fig. S1D). Together,these data revealed reciprocal regulation of AMPK and Akt

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Figure 1.

Matrix deprivation promotes Akt inactivation concomitant with AMPK activation. Representative immunoblots of the following cell lysates were probed forspecified proteins. A, Freshly isolated HMECs cultured in attached condition (Att) or as floating mammospheres (MS) in ultralow-attachment plates for 7 days.B–C, MDA-MB-231 (B) and MCF7 (C) cells cultured in attached condition (Att) or as anchorage-independent spheroids (AI-spheroids) in methylcellulose for7 days. D–E, MDA-MB-231 cells cultured in attached condition (Att) or subjected to suspension (Sus) for 8 hours on poly-HEMA-coated plates. Graphsrepresent densitometric quantification of immunoblots; error bars, mean � SEM; n ¼ 3.

Saha et al.





activities between matrix-attached and matrix-detached condi-tions, resulting in a pAkthigh/pAMPKlow state in adherentlygrowing cells, whereas a pAMPKhigh/pAktlow state in matrix-detached cells.

Akt repression in suspension is vital for anoikis resistanceIntrigued by the reduction in Akt activity in detached breast

cancer cells, contrary to its generally accepted role in anoikisresistance, we examined the relative functional roles of Akt andAMPK in detached cells. Treatment ofMDA-MB-231 cells with thePI3K inhibitor LY294002 led to a decrease in pAktS473 levels(Supplementary Fig. S2A) while treatment with AMPK inhibitorcompound C (24) led to a decrease in pACCS79 levels (Supple-mentary Fig. S2B), confirming the efficacies of these pharmaco-logic agents. Interestingly, inhibition of AMPK, but not Akt,increased anoikis as revealed by elevated levels of cleaved cas-pase-3 as well as increase in caspase-3 enzymatic activity incompound C treated cells (Fig. 2A and B). We obtained similarresults inMCF7 breast cancer cells also (Supplementary Fig. S2C).Consistent with these observations, we found that inhibition ofAMPK, but not Akt, abrogated anchorage-independent colonyformation in MDA-MB-231, MCF7, and BT474 cells (Fig. 2C;Supplementary Fig. S2D and S2E). Interestingly, our observationsin breast cancer cell lines also held true in HMEC-derived mammo-spheres, wherein again inhibition or knockdown of Akt failed toaffectmammosphere formation (Supplementary Fig. S2F and S2G).

Following this, we sought if it is necessary to maintain reducedAkt activity for stress survival in suspension. To address this, weevaluated the effects of forced Akt activation on anoikis by stablyexpressing a constitutively active HA myr-Akt construct in MDA-MB-231 cells. Detection with antibodies against HA and total Aktconfirmed exogenous protein expression (Fig. 2D). As expected,we observed increase in the levels of pAktS473 in these cells inadherent condition (Fig. 2D). To our surprise, however, whenthese cellswere subjected tomatrix deprivation,we failed to detectelevated pAktS473 levels (Fig. 2D). We obtained similar results inMCF7 cells transiently transfected with HAmyr-Akt (Supplemen-tary Fig. S2H), suggesting that myr-Akt is still susceptible tonegative regulation under matrix deprivation.

One major means of negative regulation of Akt is by dephos-phorylation (5). To circumvent this, we used constitutively active,GFP-tagged phosphomimetic HA-Akt-T308D S473D construct(25), which is refractory to the action of phosphatases, andconfirmed exogenous expression (Fig. 2E). Further, its expressionled to elevated Akt activity in adherent as well as matrix-deprivedcells, as gauged by increased levels of pPRAS40T246, a downstreamsubstrate of Akt (Fig. 2E). When these cells were subjected tosuspension for 48 hours, to our surprise, we observed increasedanoikis (Fig. 2F and G). Consistent with these data, overexpres-sion of GFP-HA-Akt-T308D S473D also impaired anchorage-independent colony formation (Fig. 2H). We obtained similarresults in MCF7 cells transiently transfected with GFP-HA-Akt-T308D S473D construct (Supplementary Fig. S2I and S2J), sug-gesting that under the stress of matrix deprivation, hyperactiva-tion of Akt might be detrimental to cell survival.

AMPK-mediated stabilization of PHLPP2 promotes Aktinactivation in suspension

We next investigated the underlying mechanisms of Akt-dephosphorylation in suspension. Because matrix deprivationled toAkt dephosphorylation concomitantwithAMPKactivation,

we investigated if AMPKmight be directly involved in this process.Interestingly, inhibition of AMPK using compound C led toelevated pAktS473 levels in MDA-MB-231 (Fig. 3A), MCF7 (Sup-plementary Fig. S3A), and BT474 (Supplementary Fig. S3B) cells.Further, overexpression of DN AMPK (Supplementary Fig. S3C)or depletion of AMPK using shRNA (Supplementary Fig. S3D)also led to elevated pAktS473 levels in suspension, together reveal-ing a direct role for AMPK in Akt dephosphorylation in matrix-deprived cells.

Our observation that matrix deprivation led to Akt dephos-phorylation in cells stably overexpressingmyr-Akt, but not in cellsstably overexpressing the double phosphomimetic Akt DDmutant (Figs. 2D and E), suggested the possible involvement ofphosphatases in this process. We, therefore, investigated the rolesof the Akt phosphatases PP2A and PHLPP. Inhibition of PP2Awith okadaic acid failed to restore Akt phosphorylation inmatrix-detached cells (Supplementary Fig. S3E). Interestingly, weobserved a significant increase in the protein levels (Fig. 3B) aswell as activity (Fig. 3C) of PHLPP2, which has specificity for Akt1(26), the major Akt isoform expressed by breast cancer cells (27),in matrix-deprived MDA-MB-231 cells. Further, knockdown ofPHLPP2 with two independent shRNA sequences led to signifi-cant increase in the levels of pAktS473 in suspension (Figs. 3D;Supplementary Fig. S3F). In addition, shPHLPP2 cells alsoshowed remarkable increase in the levels of pAktT308 in suspen-sion (Supplementary Fig. S3G). These data suggested a role forPHLPP2 in Akt inactivation in suspension.

Thereafter, we gauged if AMPK is involved in the observedupregulation of PHLPP2. In the presence of AMPK inhibitorcompound C (Fig. 3E), knockdown of AMPK (Fig. 3F) or over-expression of DN AMPK (Supplementary Fig. S3H), we observedreduced levels of PHLPP2 in suspension in MDA-MB-231 cells.Inhibition of AMPK in matrix-deprived MCF7 cells also led to adecrease in PHLPP2 levels, in parallel with an increase in pAktS473

levels (Supplementary Fig. S3A). Also, we observed decreasedlevels of PHLPP2 upon overexpression of DN AMPK in adherentMDA-MB-231 cells (Supplementary Fig. S3I) and in AMPKa�/�

MEFs (Supplementary Fig. S3J). Consistent with these observa-tions, pharmacologic activation of AMPK with A-769662 (Sup-plementary Fig. S3K), as well as genetic approach involvingconstitutively active CaMKK (Supplementary Fig. S3L), led to anincrease in the levels of PHLPP2 in adherent cells. These data thusidentified a novel positive regulation of PHLPP2 by AMPK.

We investigated potential mechanisms underlying AMPK-mediated upregulation of PHLPP2 in suspension. RT-PCR anal-yses revealed no significant change in the transcript levels ofPHLPP2 between matrix-attached and detached cells (both in thepresence and absence of AMPK inhibitor; Supplementary Fig.S4Ai). Further, we investigated the levels of miR-205, which hasbeen reported to regulate PHLPP2 levels (28). Quantitative PCRanalyses failed to detect significant change in the levels ofmiR-205between adherent and matrix-deprived conditions in MDA-MB-231 and MCF7 cells (Supplementary Fig. S4Aii). Further, inhibi-tion of AMPK also did not alter the levels of miR-205 in matrix-deprived cells (Supplementary Fig. S4Aii). Taken together, theseobservations were suggestive of possible posttranscriptional reg-ulation of PHLPP2 by AMPK. A cycloheximide chase assayrevealed a more rapid decrease in the protein levels of PHLPP2in the presence of AMPK inhibitor (Fig. 3G). We obtainedsimilar data with cells expressing shAMPKa2 (SupplementaryFig. S4B), suggesting that AMPK might promote PHLPP2 protein






stabilization. However, MG132 failed to restore protein levels ofPHLPP2 under AMPK inhibited condition (Supplementary Fig.S4C). Also, ubiquitination of PHLPP2 did not change betweenattached and matrix-deprived cells (Supplementary Fig. S4D),suggesting proteasome-independent mechanism of PHLPP2 sta-bilization. Interestingly, lysosomal inhibitors restored PHLPP2protein levels under AMPK inhibited condition (Supplementary

Fig. S4E), suggesting that AMPK might mediate PHLPP2 stabilityin detached cells by regulating lysosomal degradation.

PHLPP2 knockdown inhibits autophagy and promotes anoikisTo further understand the biological significance of AMPK-

mediated Akt inactivation through PHLPP2, we investigated theeffects of PHLPP2 knockdown in anoikis and anchorage-

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Enforced Akt activation promotes anoikis. A–C, MDA-MB-231 cells treated with DMSO, LY294002 (LY), or compound C (CC) were subjected to 48 hours ofsuspension and harvested for immunoblotting (A) and caspase-3 activity assay (B); n ¼ 3. Treated cells were subjected to colony formation in methylcellulose for15 days (C); n ¼ 4. Error bars, mean � SEM. D and E, Immunoblot analyses of MDA-MB-231 cells stably expressing control empty vector or HA myr-Akt (D),and control vector (expressing GFP) or GFP-tagged HA-Akt-T308D S473D (GFP Akt DD; E) cultured in adherent (Att) or suspension (Sus) condition; n ¼ 3.F–H, After 48 hours of suspension, MDA-MB-231 cells stably expressing control vector (expressing GFP) or GFP Akt DD were subjected to immunoblotting forcleaved caspase-3 (F) and caspase-3 activity assay (G); n ¼ 3. Cells were subjected to colony formation for 15 days (H); error bars, mean � SEM of twoexperiments with three dishes each.

Saha et al.





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AMPK promotes Akt dephosphorylation in suspension via PHLPP. A and B, Representative immunoblots of MDA-MB-231 cells treated with DMSO or compound C (CC)and subjected to suspension (Sus) for 8 hours (n ¼ 5; A), and cells grown in adherent (Att) or suspension (Sus) condition for 10 minutes and 8 hours (n ¼ 3; B).C, Phosphatase assay performed with immunoprecipitated PHLPP2; IgG was used as control; n ¼ 4. Error bars, mean � SEM. D–G, Representative immunoblotsofMDA-MB-231 cells harvested under conditions detailed below.D, Cells stably expressing nontargeting shRNA (NT) or shPHLPP2 (seq #5) and subjected to suspension(Sus) for 8 hours; n ¼ 4. E, Cells treated with DMSO or compound C (CC) were subjected to suspension (Sus) for 8 hours; n ¼ 3. F, Adherent cells stablyexpressing pTRIPZ empty vector (EV) or shAMPKa2 (seq #1) were induced with doxycycline for 48 hours, followed by suspension for 8 hours; n¼ 3. G, Adherent cellspretreated with DMSO or AMPK inhibitor (CC) were treated with cycloheximide (CHX) for 20 minutes, followed by suspension culture (Sus) for indicated time points;n ¼ 3. Graph represents quantification of PHLPP2. All values represent densitometric analyses of Western blots to quantify relative levels of specified proteins.






independent colony formation.When subjected to suspension for48 hours, we observed increased levels of cleaved caspase-3(Supplementary Fig. S5A) as well as elevated caspase-3 enzymeactivity (Fig. 4A) in shPHLPP2 (seq #5) cells as compared withcontrol cells expressing nontargeting (NT) shRNA. Similar obser-vations were made with an independent shPHLPP2 (seq #3)expressing cells (Supplementary Fig. S5B) as well as in MCF7cells (Supplementary Figs. S5C and S5D). Consistent with this,PHLPP2 knockdown cells formed significantly reduced numberof colonies in methylcellulose (Figs. 4B; Supplementary Fig. S5Eand S5F). Moreover, the effects of PHLPP2 depletion on anoikis

and anchorage-independent growth were partially reversed in thepresence of LY294002 (Fig. 4A and B; Supplementary Fig. S5A),suggesting that PHLPP2 depletion promotes anoikis and impairsanchorage-independent growth, at least in part, through Aktactivation.

Recent studies have revealed the induction of autophagy as acritical survival strategy for anoikis resistance (29). Because Aktinhibits autophagy (30) and PHLPP2 knockdown cells showedAkt activation, we investigated if impairment of autophagy isresponsible for anoikis in these cells. Compared with controlNT cells, we observed reduced AO-Red fluorescence (Fig. 4C) and

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Downregulation of PHLPP2 promotes anoikis and impairs autophagy and metastasis. A–D, MDA-MB-231 cells stably expressing nontargeting shRNA (NT) orshPHLPP2 (seq #5) were subjected to 48 hours of suspension in the presence of DMSO or LY294002 (LY) and harvested for caspase-3 activity assay (A), subjectedto soft-agar colony formation (B), and AO assay (C and D); n ¼ 3. E and F, MDA-MB-231 cells stably expressing control GFP vector or GFP-tagged HA-Akt-T308DS473D (GFP Akt DD) were subjected to suspension (Sus) for 48 hours and harvested for AO assay (E) and immunoblotting (F); n ¼ 3. G and H, Graphsrepresent number of lung (left) and liver (right) nodules following tail-vein injection of cells described below; each dot represents number of nodules in asingle mouse: G, Nontargeting shRNA (NT) or shPHLPP2 (seq #5). H, Control GFP vector or GFP-tagged HA-Akt-T308D S473D (GFP Akt DD). Error bars,mean � SEM in all experiments.

Saha et al.





decreased levels of LC3B-II (Supplementary Fig. S5A) in MDA-MB-231 shPHLPP2 cells. We obtained similar results in MCF7cells transiently transfected with shPHLPP2 construct (Supple-mentary Fig. S5G), indicating reduced autophagy upon PHLPP2depletion. Furthermore, treatment with LY294002 restoredautophagy induction in matrix-deprived shPHLPP2 cells (Fig.4D; Supplementary Fig. S5A), suggesting that elevated Akt activityin these cellsmight be one possible reason for reduced autophagy.Consistent with this notion, we observed that MDA-MB-231 cellsstably expressing GFP-HA-Akt-T308D S473D also displayedreduced AO-Red fluorescence and decreased levels of LC3B-II,indicating reduced autophagy (Fig. 4E and F). Thus, our observa-tions suggest that AMPK-mediated suppression of Akt activitypossibly facilitates anoikis resistance through facilitating theinduction of autophagy.

Because anoikis resistance contributes to metastatic potentialof cancer cells, we investigated the effects of PHLPP2 knock-down or overexpression of phosphomimetic Akt on metastasis.Tail-vein injections in nude mice revealed that MDA-MB-231cells stably expressing shPHLPP2 (Fig. 4G; Supplementary Fig.S5H) or Akt-T308D S473D (Fig. 4H; Supplementary Fig. S5I)were impaired in their metastatic potential, thus corroboratingour observations of increased anoikis and decreased anchorage-independent growth.

Adhesion-dependent double-negative feedback loop betweenAMPK and Akt

Our data thus far revealed that detachment-triggered AMPKleads to Akt inactivation, which is critical for stress survival underdetachment. We reasoned that after exiting the circulation andfollowing attachment at the new site, proliferative signals arecritical for secondary tumor growth, which might require reacti-vation of Akt and inactivation of AMPK. Therefore, we investi-gated the status of AMPK and Akt signaling in cells subjected toreattachment following matrix detachment. Reattachment ofMDA-MB-231 (Fig. 5A) and MCF7 (Supplementary Fig. S6A)cells following matrix deprivation quickly led to restoration ofpAktS473 levels comparable with originally adherent cells. Con-comitant with reattachment, we observed rapid dephosphoryla-tion of AMPK, as early as 1 hour of reattachment, in both MDA-MB-231 (Fig. 5A) and MCF7 (Supplementary Fig. S6A) cells.Further, reattachment of cells in the presence of Akt inhibitorimpaired the attachment-triggered dephosphorylation of AMPKin both MDA-MB-231 (Fig. 5B) and MCF7 (Supplementary Fig.S6B) cells, suggesting a direct role for Akt in AMPK dephosphor-ylation following adhesion.

We sought to investigate the role of phosphatases in theadhesion-mediated dephosphorylation of AMPK. We did notdetect any differences in the levels or activities of PP2A-Aa/b,PPM1E, and themonomeric phosphatase PP2C-a, that have beenidentified as cellular AMPK phosphatases, between adhered anddetached conditions (Supplementary Fig. S6C and S6D). Inimmunoprecipitation experiments using individual phospha-tase-specific antibodies, we observed no change in the interactionbetween AMPK and PP2A-Aa/b or PPM1E between adherent anddetached cells (Supplementary Fig. S6E and S6F). Interestingly,we observed a noticeable increase in the interaction betweenAMPK and PP2C-a under adhesion compared with detachment(Fig. 5C). A reverse pulldown using AMPKa2-specific antibodiesfurther confirmed these results (Supplementary Fig. S6G).Consistent with this, PP2C-a knockdown led to increased

pAMPKaT172 levels in adherent cells (Fig. 5D). Further, inhibitionof Akt, while it did not cause a change in PP2C-a activity(Supplementary Fig. S6H), impaired the interaction betweenAMPK and PP2C-a in adherent cells (Fig. 5E), together suggestingan Akt-dependent, PP2C-a–mediated dephosphorylation ofAMPK in adhesion.

Thus, our data showed that while matrix detachment-triggeredAMPK activation inhibits Akt, attachment-triggered Akt activationinhibits AMPK. These data are suggestive of a double-negativefeedback loop between these two kinases in matrix-adheredversus matrix-detached states of cells. To further confirm this, wetested the effect of forced activation of AMPK on pAktS473 levels inadherent conditions (where normally AMPK activity is low) andforced activation of Akt on pAMPKaT172 levels in suspension(where normally Akt activity is low). Activation of AMPK inadherent conditions, mediated by AMPK activator A-769662 (Fig.5F; Supplementary Fig. S7A), overexpression of constitutivelyactive AMPK-upstream kinase CaMKK (Fig. 5G; SupplementaryFig. S7B), or knockdown of PP2C-a (Fig. 5H; Supplementary Fig.S7C), promoted Akt inactivation. On the other hand, forcedactivation of Akt in suspension, as observed in cells stably expres-sing phosphomimetic Akt-T308D S473D (Fig. 2E) or shPHLPP2(Fig. 3D), promoted AMPK inactivation (Fig. 5I and J; Supple-mentary Fig. S7D and S7E). Collectively, these data identify anovel double-negative feedback loop between the two cellularkinases Akt and AMPK, which maintains an attachment-triggeredpAkthigh/pAMPKlow state in adherent cells while maintaininga detachment-triggered pAMPKhigh/pAktlow state in matrix-deprived cells.

To further corroborate the double-negative cross-talk betweenAMPK and Akt a microarray-based gene expression analysis wasperformed between adherent and detached MDA-MB-231 cells.Consistent with higher levels of pAkt in adherent cells, weobserved elevated Akt gene signature, including upregulation ofAKT1, CDC34, NEDD8, PRKCD, and DUSP10 (31) in adherentcondition. Further, in keeping with a role for Akt signaling inanabolic pathways, we observed elevated expression of genesinvolved in fatty acid synthesis (such as ACAA1 and 2, ACAD10 and 11, ACOX 1 and 2, ACSBG 1 and 2, and ACSL 3, 5, and 6)and pentose phosphate pathway (such as G6PD, H6PD, PGLS,PRPS1, and PRPS1L1; ref. 32). Additionally, we observed elevatedexpression of genes involved in the mTOR pathway and proteinsynthesis (such as EIF4B, HIF1A, RPS6, EIF4EBP1, EIF4EBP2,PPP2CA, RPS6, and IKBKB; ref. 31) and in cell growth andproliferation (such as ANAPC2, CDK4, CDK6, CDKN3, CUL1,E2F1, SKP2, CDC6, and WEE1; ref. 33) in adherent cells (Fig. 6Aand B).

Similarly, AMPK activation in matrix-detached cells wassupported by the upregulation of AMPK gene signature, includ-ing PTPLB, SFXN1, FKBP5, TUSC5, ORM1, CEBP1, MAP3K6,and PRKAR2B (Fig. 6A; refs. 18 and 34). Additionally, genesinvolved in catabolic processes including fatty acid oxidation(such as HSD17B4, ACOT8, ACOX3, ABCD1, and ACOX1;ref. 32), and glycolysis (such as ALDOA, ALDOC, ENO1,2,3,GALM, HK2, G6PD, and ALDOB; ref. 32), and genes involved instress-responsive pathways including autophagy, oxidativestress, and hypoxia (such as ATG4, ATG9, GABARAPL1, IRGM,MAP1LC3A, and ULK1; ref. 35) were elevated in detachedMDA-MB-231 cells (Fig. 6C).

Moreover, AMPK knockdown reversed the expression of severalof these genes inmatrix-detached cells (Fig. 6A–C), revealing their






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Matrix reattachment leads to Akt-dependent repression of AMPK activity. A–E, Representative immunoblots of cells harvested under conditions detailed below.A, MDA-MB-231 cells cultured in attachment (Att), suspension for 10 minutes (Sus), or allowed to reattach (Re-att); n ¼ 3. B, MDA-MB-231 cells subjected to10 minutes of suspension (Sus) were allowed to reattach (Re-att) in the presence of DMSO or Akt inhibitor; n¼ 3. C, Lysates of MCF7 cells cultured in conditions ofattachment (Att), suspension (Sus), and reattachment (Re-att) were immunoprecipitated (IP) with control IgG or anti–PP2C-a antibodies and analyzed byimmunoblotting. The input represents 2% of the whole-cell lysate used for each immunoprecipitation; n¼ 3. D, Adherent MCF7 cells transfected with vector control(pLKO.1) or shPP2C-a; n ¼ 3. E, Lysates of adherent MCF7 cells treated with DMSO or Akt inhibitor were immunoprecipitated and analyzed as describedin C; n ¼ 3. F–J, Graphs represent densitometric quantification of immunoblots (Supplementary Figs. S7A-S7E) for relative phospho-protein levels from cellsharvested under conditions detailed below: F, Adherent MDA-MB-231 cells treated with DMSO or AMPK activator (A76; also see Supplementary Fig. S7A); n ¼ 3.G, Adherent MDA-MB-231 cells transfected with control vector pEGFP or GFP-tagged constitutively active CaMKK (GFP CA CaMKK; also see SupplementaryFig. S7B); n¼ 3.H, AdherentMCF7 cells transfectedwith vector control (pLKO.1) or shPP2C-a (also see Supplementary Fig. S7C); n¼ 2. I, MCF7 cells transfectedwithGFP or GFP-HA-Akt-T308D S473D and subjected to suspension (8 hours; also see Supplementary Fig. S7D); n ¼ 2. J, MDA-MB-231 cells stably expressingnontargeting shRNA (NT) or shPHLPP2 (seq #5) and subjected to suspension (48 hours; also see Supplementary Fig. S7E); n ¼ 3. Error bars, mean � SEM.

Saha et al.





AKT1CDC34

MVKNEDD8PRKCDUBE2MDUSP10PMPCA

ADIPOR1KCTD5

TOLLIPRNF126

DKK1JAG1

JunBIK

BMPERNOS3

ANGPT2BMP2CDH2

FSTCXCL12

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GRB10GRB2

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PIK3CGPIK3R1

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PTENTCL1AHSPB1

ILKBTK

PDK2GPX4

LASP1MMP15

CLSTN1BRMS1

TJP3FGF2

IGFBP2BMP4

AngIGFBP3

IL6DLL1DLL4JAG2

Csf2Traf1Trib3Cd69Cd52

Gata3Egr1

SLC36A2ACP5HPCYP2E1MCF2LAOC3CFDADIPOQAACSSLC7A8SLC25A1PPARGARRDC2DGAT2FMO2CHDHSLC7A10PPP1R3CITGB1BP3FOXO1PCK1UCP1CDO1

ADAMTSL4ANKRD2TIMP4XDHNNATCSRP3

TMEM45BLMOD2MYL2SYP

PTPLBSFXN1FKBP5TUSC5ORM1RETNCIDECPIK3CDFABP4ADRB3IER5LEPCEBPAAGPAT2FASNMGST1TKTPYGLELOVL6MAP3K6CIDEAACLYTHRSPLIPEPRKAR2BSLC1A5

Akt pathway AMPK pathway

CDKN1BCUL3ABL1

SERTAD1CDC25CSTMN1

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ANAPC2CCND1CCNE1

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CDKN3CUL1CUL2E2F1SKP2CDC6WEE1

ANAPC2CDC25ACDK5R1

CDK7CDKN3CKS1B

CKS2GTSE1KPNA2MNAT1AURKBCCNB2

CCNFCDC6

CDC16CDC20

MRE11ARAD51BRCA1CASP3RBL1

AMBRA1ATG16L1ATG4BATG4DATG9AATG9BGABARAPL1GABARAPL2IRGMMAP1LC3AULK1WIPI1DRAM1LAMP1NPC1

ATG12ATG4AATG4CATG5GABARAPMAP1LC3BRGS19GABARAP

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proliferation

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BNIP3CYP1A1GPX3PRDX2ATMGADD45GTP53XPCCSTBRARADNAJC15PRDX2COX2SCARA3BRCA1CCND1GADD45AMLH1MSH2INSIG1UBE2G2

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Microarray-based expression profiling of genes regulated in matrix-attached versus matrix-detached states of cells. A–D, Heat map depicting unsupervisedclustering of gene expression profiles from MDA-MB-231 cells cultured in adherent condition (Att), suspension for 24 hours (Sus), and expressing shAMPKa2.Genes of interest are color coded. Red, highly expressed genes; green, downregulated genes. The zoomed section of the heat map represents genes involved in Aktand AMPK pathways (A), cell growth, proliferation, autophagy, and cellular stress conditions (B), metabolic processes (C), and cell–cell and cell–ECM adhesions (D).E, Heat map depicting semisupervised clustering of genes involved in Akt signaling across primary breast tumor, CTCs, and metastases. F, Box plots showdistribution of normalized (Z-score) gene expression of AMPK-dependent genes frommicroarray data available for primary tumor (GSE43837), CTCs (GSE99394),and metastases of breast cancer patients (GSE43837 and GSE56493).






AMPK dependency. We also observed AMPK-dependent alteredexpression of integrins that are known to be involved in anoikisresistance and contribute tometastasis (36) in detached cells (Fig.6D). In addition, microarray data analyses of matrix-detachedPHLPP2-knockdown cells and GFP-HA-Akt-T308D S473D-expressing cells also exhibited upregulation of the Akt pathwayand apoptotic genes while showing downregulation of autop-hagy-related genes (Supplementary Fig. S7F–S7H), thus support-ing our experimental data. Collectively, these data supported theconcept of double-negative feedback loopbetweenAkt andAMPKin matrix-adhered versus detached states of cells.

In order to understand the functional relevance of the double-negative feedback between Akt and AMPK in breast cancer pro-gression, we examined publicly available microarray data fromthe GEO of patient-derived primary breast tumors (GSE43837),circulating tumor cells (GSE99394), and metastatic lesions atdifferent organs (GSE43837 and GSE56493). Similar to adher-ently growing MDA-MB-231 cells (Fig. 6 A–D), heat map gener-ated from cDNA microarrays of patient samples revealed an Aktpathway associated gene expression pattern in primary and met-astatic lesions (Fig. 6E) that was suppressed in CTCs. In contrast,and similar to detachedMDA-MB-231 cells (Fig. 6B), we observedAMPK-dependent gene expression, including those of stress sig-naling and autophagy, in patient-derived CTC microarray datathat were suppressed in primary and metastatic lesions (Fig. 6F).For example, box plot analysis showed AMPK-dependent upre-gulationofACLY, RETN (18), and autophagy-related genesNPC1,LC3B, CYP1A1, BRCA1, andUBE2G2 (37), while revealing down-regulation ofNNAT, ANKRD2, and LIPE (Fig. 6F; ref. 18) in CTCs.

DiscussionOnce tumorsmetastasize to a distal site, they aremostly fatal to

the patient due to lack of strategies currently to treat metastasis.Therefore, understanding the molecular mechanisms that con-tribute to cancer metastasis can advance treatment approach.Tumor cells grow adherently both in primary and secondarytumor sites, but they need to overcome the stress of matrix

deprivation during transit through the circulation. Yet, whatmaintains the states of adhesion, characterized by cell growthand proliferation, versus the state of matrix-detachment, charac-terized by stress survival, remains poorly understood. In thisstudy, we show that AMPK activation triggered by matrix depri-vation leads to a concomitant inactivation of Akt by stabilizingPHLPP2 in breast cancer cells. Knockdown of PHLPP2 or consti-tutive activation of Akt increased anoikis while impairing autop-hagy, thus inhibiting anchorage-independent growth and metas-tasis, and highlighting the importance of suppression of Aktactivity for surviving the stress of matrix deprivation. We furtherdemonstrate that matrix reattachment-triggered Akt activationconcomitantly promotes AMPK inactivation through yet anotherphosphatase PP2C-a. Our data, thus, reveal for the first time areversible, double-negative feedback loopbetweenAMPKandAktbetween matrix-adhered versus detached states that can coordi-nate the intracellular signaling pathways involved in cell growthand stress survival during cancer progression.

Akt signaling represents a widely accepted prosurvival pathwaythat is known to aid anoikis resistance and tumor progression(38). Surprisingly, we observed Akt repression concomitant withAMPK activation in a large array of matrix-deprived epithelialcancer cells (of lung, glial, cervix, liver origin) including breastcancer cell lines MDA-MB-231 and MCF7. This prompted us toinvestigate if suppression of Akt activity in suspension is requiredfor cell survival. To address this, we enforced Akt activation inmatrix-deprived cells using constitutively active Akt constructs.Interestingly, even though both myr-Akt and the phosphomi-metic mutant Akt-T308D S473D led to elevated Akt activity inadherent cells, we detected increased Akt activity in suspensiononly with the expression of Akt-T308D S473D, but not with myr-Akt, an oft-used construct throughout literature. Based on ourfindings that matrix deprivation upregulates the Akt phosphatasePHLPP2, we predict that myristoylated, membrane-tetheredforms of Akt are still susceptible to negative regulation in sus-pension by phosphatases. Interestingly, expression of Akt-T308DS473D, which is refractory to dephosphorylation at the keyactivating phosphorylation sites, promoted anoikis, suggesting

MATRIX-ATTACHEDpAkthigh/pAMPKlow

Akt/mTOR pathway

Anabolic state

Fatty acid biosynthesis

Protein translation

Cell cycle

Cell growth and proliferation

AMPK

Akt

PP2C-α

MATRIX-DEPRIVED

pAMPKhigh/pAktlow

AMPK pathway

Catabolic state

Fatty acid oxidation

Glycolysis

Autophagy

Stress-survival

Akt

AMPKPHLPP2

Detachment

Attachment

Figure 7.

Amodel for double-negative feedbackloop between two cellular kinasesAMPK and Akt. We show thatdetachment-triggered AMPKconcomitantly inactivates Akt throughthe phosphatase PHLPP2, resulting ina pAMPKhigh/pAktlow catabolic statethat facilitates stress-survival inmatrix-deprived cells. In contrast,adhesion-triggered Akt keeps AMPKunder check by the phosphatasePP2C-a, maintaining a pAkthigh/pAMPKlow anabolic state, which isconducive for cell growth andproliferation. Thus, betweenattachment and detachment, AMPKand Akt constitute a reversibledouble-negative feedback loop,maintaining stable pAkthigh/pAMPKlow

and pAMPKhigh/pAktlow states, yetretaining the ability to switch betweenthese two cellular states.


Saha et al.




that high Akt activity might be detrimental to cancer cell survivalin suspension. This is in agreement with yet another report thatshowed elevated Akt activation leads to cell death due to increasein reactive oxygen species (39).

Our data revealed AMPK-dependent upregulation of PHLPP2in suspension. However, we failed to detect changes in transcriptlevels of PHLPP2, suggesting possible posttranscriptional regula-tion. Although PHLPP2 has been shown to be targeted forubiquitin-mediated degradation (40), we failed to see changesin its ubiquitination or effect of inhibition of proteasomal deg-radation. Instead, our data suggested a possible role for lysosomaldegradation in regulating PHLPP2 protein levels. Interestingly, arecent paper has demonstrated a role for PHLPP1 and Akt in theregulation of chaperone-mediated autophagy that regulates pro-tein degradation (41). However, a selective chaperone-dependenttargeting of PHLPP2 for lysosomal degradation remains to beexplored. Yet another possible mechanism of PHLPP2 upregula-tion could involve rapid protein synthesis aswe observed elevatedPHLPP2 levels within 10 minutes of suspension. Because AMPKactivation is known to inhibit cap-dependent translation insuspension (18), this might possibly involve stress-associatedalternate modes of translation.

Reduced PHLPP2 protein expression in colon cancer andpancreatic ductal adenocarcinoma patient samples, and anti-tumorigenic effects of its overexpression in colon and pancreaticcancer cell lines (42, 43) have largely supported tumor-suppres-sive functions for PHLPP2. In contrast, our results showed thatdepletion of PHLPP2 rendered breast cancer cells anoikis-sensi-tive and impaired metastasis. In support of this, a recent studyusing PTEN/TP53-mutant prostate cancer mice showed that mycdrives proliferation andmetastasis through activation of PHLPP2and suppression of the Akt pathway (44). Together, these findingsbegin to highlight novel, context-specific tumorigenic functionsfor PHLPP2.

We report here an AMPK-mediated suppression of Akt activityinmatrix-deprived cell survival. We speculate that such a negativeregulation of Akt by AMPKmight be favorable tomatrix-deprivedcells because this can shift cellular signaling from energy-con-suming/anabolic processes (mediated by Akt activation) to ener-gyproducing/catabolic processes (mediatedbyAMPKactivation),thus restoring energy homeostasis. This is consistent with a recentfinding revealing AMPK-mediated inhibition of mTOR and sup-pression of protein synthesis in anoikis resistance (18). Further,the catabolic process of autophagy, which is also known toregulate energy balance, plays a key role in anoikis-resistance(29). Interestingly, Akt activation has been shown to inhibitautophagy (30). In keeping with this, our present data showedthat PHLPP2 knockdown or enforced Akt activation in matrix-deprived cells inhibited autophagy and increased anoikis. Thus,our data suggest that AMPK-mediated Akt inactivation mightadditionally contribute to anoikis resistance by promotingautophagy.

Matrix attachment leads to Akt activation through integrinsignaling (45), while we have identified elevated calcium andROS levels as triggers for detachment-induced AMPK activation(46). We show here that reattachment to the matrix restores Akt

activity leading to AMPK inactivation through increased interac-tion between AMPK and its phosphatase, PP2C-a. While PP2C-ais predominantly nuclear localized (47), AMPK is reported toshownuclear localization and cytoplasmic redistribution throughRan-dependent import and CRM1-mediated export pathways(48). Interestingly, Akt has been shown to regulate RanBP andCRM1-dependent nuclear-cytoplasmic shuttling of proteins (49).Thus, one possible way by which Akt could regulate the interac-tion between AMPK and PP2C-a might involve Akt-dependentredistribution of AMPK.

These findings, thus, identify a novel, and reversible, double-negative feedback loop between AMPK and Akt between matrix-attached and matrix-deprived conditions. Comparison of micro-array gene expression data of primary tumors, circulating tumorcells, and metastatic lesions further suggested that such a recip-rocal regulation might exist in metastatic breast cancers. Wepropose that such a reversible regulationmight help rapid switch-ing between pAkthigh/pAMPKlow state in adhesion that favors cellgrowth, to pAMPKhigh/pAktlow state that allows adaptation tomatrix-detachment stress (Fig. 7), yet retaining the ability to restoregrowth following proper attachment at the secondary site. Disrupt-ing this loop using AMPK or PHLPP2 inhibitors might providenovel therapeutic strategies to restrict metastatic cancer spread.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Saha, S. Kumar, S.K. Hindupur, A. RangarajanDevelopment of methodology: M. Saha, S. Kumar, S. Bukhari, S.A. Balaji,S.K. HindupurAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Saha, S. Kumar, S.A. BalajiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Saha, S. Kumar, S. Bukhari, P. Kumar,S.K. Hindupur, A. RangarajanWriting, review, and/or revision of the manuscript: M. Saha, S. Kumar,S. Bukhari, P. Kumar, S.K. Hindupur, A. RangarajanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Saha, A. RangarajanStudy supervision: A. Rangarajan

AcknowledgmentsThis work was supported by the Wellcome Trust/DBT India Alliance Fel-

lowship (grant number 500112-Z-09-Z) awarded to A. Rangarajan. Grants fromDBT-IISc partnership program and support from DST-FIST and UGC, Govern-ment of India, to the Department of MRDG are also acknowledged.

The authors thankDrs.M. Vijaya Kumar andRekha V. Kumar for their help inprocuring primary tissue samples at KMIO; Drs Benoit Viollet for AMPK DKOcells and Deepak Saini for shRNA of phosphatases; Sukrutha Reddy, NehanjaliDwivedi, and Sunaina Rao for help with immunoblotting; and Srikanth S.Manda, Institute of Bioinformatics, for analysis of microarray data. The authorsacknowledge the Central Animal Facility and FACS facility at IISc.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 17, 2017; revised November 17, 2017; accepted January 10,2018; published OnlineFirst January 16, 2018.

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2018;78:1497-1510. Published OnlineFirst January 16, 2018.Cancer Res Manipa Saha, Saurav Kumar, Shoiab Bukhari, et al. Regulates Their Adaptation to Matrix Deprivation

Akt Double-Negative Feedback Loop in Breast Cancer Cells−AMPK

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