AMPK–Akt Double-Negative Feedback Loop in Breast Cancer Cells Regulates Their Adaptation to Matrix Deprivation Manipa Saha1, Saurav Kumar1, Shoiab Bukhari1, Sai A

Published OnlineFirst January 16, 2018; DOI: 10.1158/0008-5472.CAN-17-2090

Cancer Tumor Biology and Immunology Research

AMPK–Akt Double-Negative Feedback Loop in Breast Cancer Cells Regulates Their Adaptation to Matrix Deprivation Manipa 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- of the pAkthigh/pAMPKlow state. Clinical specimens of primary kis. Disseminated tumor cells must adapt to survive matrix and metastatic breast cancer displayed an Akt-associated gene deprivation, while still retaining the ability to attach at sec- expression signature, whereas circulating breast tumor cells ondary sites and reinitiate cell division. In this study, we displayed an elevated AMPK-dependent gene expression signa- elucidatemechanismsthatenablereversiblematrixattachment ture. Our work establishes a double-negative feedback loop by breast cancer cells. Matrix deprival triggered AMPK activity between Akt and AMPK to control the switch between matrix- and concomitantly inhibited AKT activity by upregulating the attached and matrix-detached states needed to coordinate cell Akt phosphatase PHLPP2. The resultant pAMPKhigh/pAktlow growth and survival during metastasis. state was critical for cell survival in suspension, as PHLPP2 Signiﬁcance: These ﬁndings reveal a molecular switch that silencing also increased anoikis while impairing autophagy regulates cancer cell survival during metastatic dissemination, and metastasis. In contrast, matrix reattachment led to Akt- with the potential to identify targets to prevent metastasis in mediated AMPK inactivation via PP2C-a-mediated restoration breast cancer. Cancer Res; 78(6); 1497–510. 2018 AACR.

Introduction vival, and metabolism, and plays a major role in tumor progression (4). Akt is recruited to the plasma membrane by binding to Metastasis accounts for the vast majority of cancer-associated PIP3 and is subsequently phosphorylated by PDK1 and mTOR deaths. The metastatic process involves detachment of cells from complex 2 (mTORC2) at T308 and S473, respectively, leading to the primary site of tumor initiation, entry into the blood stream or its full activation. Conversely, Akt signaling is attenuated by the lymphatics, exit from the circulation and reattachment at dephosphorylation of these sites by protein phosphatase 2A distant sites to spawn metastatic growth (1). Integrins mediate (PP2A) and pleckstrin homology domain leucine-rich repeat cell adhesion to the extracellular matrix that provides growth and protein phosphatases (PHLPP 1 and 2; ref. 5). Upon activation survival signals (2), whereas matrix deprivation leads to pro- by growth factor signaling, Akt promotes anabolic processes grammed cell death termed "anoikis" (3). Therefore, detached including lipid biosynthesis and protein translation, thus driving tumor cells must develop resistance to anoikis, while retaining the cell growth and proliferation. ability to reattach and grow at a distal site to spawn a successful In contrast, the AMP-activated protein kinase (AMPK) is acti- metastasis. Yet, little is known about cellular signaling pathways vated under metabolically stressed conditions and brings about that coordinate cell growth and stress-survival signals during the cellular homeostasis by switching on energy-generating catabolic attachment–detachment cascade of metastatic colonization. processes like fatty acid oxidation and glycolysis, while inhibiting The serine/threonine protein kinase Akt (also known as PKB) energy-consuming anabolic pathways including carbohydrate, regulates several cellular processes, including proliferation, sur- lipid, and protein biosynthesis (6–8). AMPK is a heterotrimeric protein consisting of a, b, and g subunits (encoded by a1, a2; b1, b2; and g1, g2, g3). It is allosterically activated by AMP and 1Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India. 2Institute of Bioinformatics, International positively regulated by phosphorylation of T172 residue by Technology Park, Whitefield, Bangalore, India. upstream kinases LKB1 and CaMKKb, while negatively regulated by dephosphorylation (9, 10). Although considered a tumor Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). suppressor owing to its growth retarding effects, recent studies have identified context specific protumorigenic roles for AMPK by M. Saha and S. Kumar contributed equally to this article. promoting cell survival under glucose deprivation and hypoxia Current address for Sravanth K. Hindupur: Biozentrum, University of Basel, stress (11, 12). Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland. Under matrix-deprivation stress, Akt activation is sufficient for Corresponding Author: Annapoorni Rangarajan, Indian Institute of Science, Lab anoikis resistance in immortalized MDCK cells (13). ErbB2-over- GA 10 MRDG IISc Bangalore, Bangalore 560012, Karnataka, India. Phone: 91-80- expressing breast cancer cells show increased dependence on Akt 22933263; E-mail: [email protected] for anchorage-independent growth (14). In contrast, pharmaco- doi: 10.1158/0008-5472.CAN-17-2090 logic inhibition of the PI3K/Akt pathway failed to render T-47D 2018 American Association for Cancer Research. breast cancer cells sensitive to anoikis (15). Thus, the role of Akt in

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anoikis resistance remains to be fully understood. On the other respectively, as kind gifts. shRNAs against PHLPP2 (RHS4531- hand, recent work from our laboratory and that of others has EG23035) and the corresponding control nontargeting shRNA in shown matrix deprivation-triggered activation of AMPK and its pGIPZ vector (NT); and inducible shRNA against AMPKa2 critical role in anoikis resistance in breast cancer cells (16–18). (V2THS_57674) and the corresponding control empty pTRIPZ Thus, independent studies have implicated Akt and AMPK in vector (EV) were procured from Dharmacon. Lipofectamine anoikis resistance, although they have opposing effects on cellular (Invitrogen) was used to transfect plasmid DNA into cells. growth and metabolism. MDA-MB-231 cells stably expressing GFP-HA-Akt-T308D Synergistic and antagonistic relationship between Akt and S473D were generated by transfection followed by FACS-based AMPK has been documented under different cellular contexts; sorting for GFP-expressing cells; cells stably expressing HA myr- however, little is known about their interplay in maintaining the Akt were generated by cotransfecting a puromycin resistance adherent versus detached states of cells. Intriguingly, we show plasmid at a 10:1 ratio followed by selection with puromycin here that detachment-triggered AMPK concomitantly represses (0.5 mg/mL) treatment. MDA-MB-231 cells stably expressing Akt activity. We identify a novel AMPK-mediated PHLPP2 upre- speciﬁc shRNAs were generated by selection with puromycin gulation that inactivates Akt to promote AMPK-induced autop- followed by sorting cells for high GFP (in case of plasmids in hagy and that inhibits anoikis in suspension. Finally, we show that pGIPZ vector) or high RFP (in case of plasmids in pTRIPZ vector) matrix reattachment triggers Akt activity, which in turn represses expression. AMPK through PP2C-a. Our data, thus, identify a novel, recip- siRNA oligos against Akt (targeting both isoforms Akt1 and rocal, inhibitory relationship between AMPK and Akt that reg- Akt2 [6211 and 6510]) were purchased from Cell Signaling ulates adaptation to matrix detachment. Technology and transfected using oligofectamine (Invitrogen).

Materials and Methods Pharmacologic compounds Pharmacologic compounds used in cell culture include the Primary cells and culture conditions AMPK inhibitor 6-[4-(2-piperidin-1-ylethoxy-phenyl)]-3-pyridin- Primary breast tissues (cancer and adjacent normal) obtained 4-yl-pyrrazolo [1, 5-a]-pyrimidine (compound C; Cat. No. 171260; from the Kidwai Memorial Institute of Oncology (KMIO), Ban- 10 mmol/L; referred to as CC in figures), PI3K/Akt inhibitor galore, as per IRB and in compliance with ethical guidelines of LY294002 (Cat. No. 440202; 20 mmol/L; referred to as LY in KMIO and the Indian Institute of Science (IISc), were processed figures), Akt inhibitor Akti VIII (Cat. No. 124018; 10 mmol/L), and into single cells and cultured as described previously (16, 19) in MG132 (Cat. No. 474790; 10 mmol/L) from Calbiochem (Merck), serum-free media containing 10 ng/mL hEGF, 1 mg/mL hydro- AMPK activator A-769662 (100 mmol/L; referred to as A76 in cortisone, 10 mg/mL insulin, 4 ng/mL heparin and B27. Single figures) from the University of Dundee, Scotland, cycloheximide cells were seeded in regular TC plates for adherent culture or in (Cat. No. C7698; 0.1 mg/mL; referred to as CHX) and lysosomal ultralow attachment plates (Corning Inc.) for mammosphere inhibitor chloroquine (Cat. No. C6628; 50 mmol/L; referred to as culture (16). CQ in figures) from Sigma-Aldrich. Dimethyl sulfoxide (DMSO) was used as vehicle control for all compounds except cyclohexi- Cell lines and cell culture conditions mide, which was dissolved in water. In experiments carried out in Breast cancer cell lines MDA-MB-231, MCF7, BT474 (from suspension, adherent cells were pretreated with the respective ATCC in 2016, and validated by STR analysis); A549 and H460 chemicals for 2 hours prior to being subjected to suspension in (lung), LN229 (glioma), Hep3B (liver), and HeLa (cervical) the continued presence of the chemicals. cancer cell lines (obtained as kind gifts) were cultured in DMEM (Sigma-Aldrich) supplemented with 10% FBS containing peni- Immunoblotting and immunoprecipitation cillin and streptomycin, at 37 C and 5% CO2. Cell lines were used For immunoblotting, whole-cell lysates were prepared using for experiments within 8 passages after thawing. For short-term lysis buffer containing 50 mmol/L Tris, 50 mmol/L sodium suspension cultures of 8 hours and 10 minutes, cancer cells were fluoride, 5 mmol/L sodium pyrophosphate, 1 mmol/L EDTA, seeded on dishes coated with 1 mg/mL poly-2-hydroxyethyl- 1 mmol/L EGTA, 1% Triton X-100, 0.2 mg/mL DTT, 0.2 mg/mL methacrylate (Poly-HEMA; Sigma-Aldrich) dissolved in absolute benzamidine, and protease inhibitor (Roche) on ice. Protein ethanol. For 48 hours of suspension culture, cells were seeded on concentration was estimated using Bradford method, equal quan- dishes coated with 1% noble agar (Sigma-Aldrich). Long-term tity of protein (30–50 mg) per lane was resolved by SDS-PAGE anchorage-independent (AI) colony formation assay was under- after boiling with sample buffer for 3 minutes at 100C. Proteins taken by admixing 1 105 cancer cells either with a slurry of 1.5% were transferred to PVDF membrane and probed with appropriate methyl cellulose or in 0.3% soft agar, and layered over 0.6% noble antibodies. The membrane was incubated overnight with agar. AI colonies from methyl cellulose were harvested for immu- primary antibody at 4C followed by washes in TBST, and incu- noblotting after 7 days or counted after 15 days from 15 random bated for 2 hours with HRP-conjugated secondary antibody at fields of 10 magnification in each 35-mm dish. room temperature. Chemiluminescence (using ECL substrate from Thermo Fisher Scientific) was used to visualize protein Plasmids, transfection, and generation of stable cell lines bands. The membranes were stripped using 1 mol/L Tris GFP-HA-Akt-T308D S473D (#39536; originally submitted by buffer (pH 6.8) containing 2% sodium dodecyl sulfate and Dr. Julian Downward), referred to as GFP Akt DD in figures, and 0.7% b-mercaptoethanol and then used for repeated probing myc AMPKa2 K45R (#15992; originally submitted by Dr. Morris with subsequent antibodies, following washes and blocking. Birnbaum), referred to as dominant-negative (DN) AMPK, were Multipanel blots were assembled by reprobing the same procured from Addgene. HA myr-Akt and GFP CA CaMKK were blot for successive antibodies or by running the same lysate provided by Dr. Joseph Testa and Dr. Grahame D. Hardie, multiple times; a-tubulin served as loading control for each run.

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Representative immunoblots show data consistent with minimally 500 mg of total protein lysate prepared from cells under attached three independent experiments. Densitometric analyses of Western (Att), suspension (Sus), and reattached (Re-Att) conditions was blots were performed using the Multigauge V2.3 software. Rela- independently immunoprecipitated with antibodies against tive protein levels were quantified by normalizing to loading PP2A-Aa/b, PPM1E, or PP2C-a and the activity of the phospha- control. Primary antibodies used in the study were against tases was estimated colorimetrically by measuring the released pAMPKaT172, pACCS79, pAktS473, pAktT308, pPRAS40T246, total phosphate from threonine phosphopeptide (K-R-pT-I-R-R) with AMPKa (that recognizes both AMPKa 1 and 2 isoforms), Malachite Green Phosphate detection solution (absorbance mea- AMPKa2, ACC, Akt, PRAS40, PP2C-a, myc tag, HA tag, cleaved sured at 640 nm). caspase-3, LC3B, GFP, Ubiquitin (Ub), IgG (Cell Signaling Tech- nology), a Tubulin (Calbiochem), PHLPP2 (Abcam), PP2A-Aa/ Microarray and data analysis b, and PPM1E (Santa Cruz Biotechnology). HRP-conjugated MDA-MB-231 cells cultured in attached or suspension condi- anti-mouse and anti-rabbit antibodies were obtained from Jack- tions for 24 hours, and MDA-MB-231 cells stably expressing son ImmunoResearch Laboratories. For immunoprecipitation shAMPKa2, shPHLPP2, and GFP Akt DD cultured in suspension experiments, cells were lysed in buffer containing 20 mmol/L were harvested for microarray experiments. Total cellular RNA was Tris (pH 8), 137 mmol/L NaCl, 10% glycerol, and 1% Nonidet P- isolated using an RNeasy minikit (Cat No.74104; Qiagen) accord- 40, supplemented with protease inhibitors, sodium fluoride, and ing to the manufacturer's protocol. The RNA samples were labeled sodium orthovanadate. Cellular protein (1 mg) was incubated withCy3 using an Agilent's Quick-Amp labeling Kit (Cat. No. 5190- with one of control IgG, anti-PP2A-Aa/b, anti-PPM1E, or anti- 0442) and subjected to hybridization on Agilent's In situ Hybrid- PP2C-a antibody and 15 mL of protein-A sepharose beads for 12 ization Kit (Cat No.5188-5242). For analysis, a list of signature hours at 4C on end-on rocker. The immune complexes were genes was taken from AmiGO Gene Ontology Consortium (http:// precipitated by centrifugation at 1300 rpm for 5 minutes at 4C. amigo.geneontology.org/amigo), Profiler PCR Array list Qiagen, The precipitates were washed with Nonidet P-40 lysis buffer sup- and further validated by KEGG (http://www.genome.jp/kegg/path plemented with 1 mol/L NaCl. Immune complexes were resus- way.html) or PubMed (https://www.ncbi.nlm.nih.gov/pubmed/). pended in 50 mL of sample buffer and analyzed by immunoblotting. Heat map of individual and combined data sets was generated using an online tool Morpheus (https://software.broadinstitute. Caspase-3 activity assay org/morpheus/#). Unsupervised clustering was performed to Caspase-3 activity was measured by using a CaspGLOW Red obtain function based gene expression signature. Genes of interest Active caspase-3 activity kit from Bio Vision (K-193) as per the are color coded; "red" in the heat map represents upregulatedgenes, manufacturer's instructions. Briefly, 1 106 cells were stained whereas "green" represents downregulated genes. with 1 mL of Red-DEVD-FMK for 30 minutes at 37 C and 5% CO2. Microarray raw data for primary breast tumor, circulating Cells were washed thrice using wash buffer and analysis was done tumor cells (CTC), and metastases were taken from Gene Expres- using BD FACS-CantoII (Becton & Dickinson) equipped with a sion Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/), with 488-nm Coherent Sapphire Solid State laser. Red (564–606 nm) GEO IDs GSE43837, GSE99394, and GSE56493. These raw data 4 fluorescence emission from 10 cells was measured after illumi- files were processed and expression values were calculated in log2 nation with blue (488 nm) excitation light. Data were analyzed scale. Further, the data were normalized using Z-score. Microarray using Summit software V5.2.1.12465. for GSE43837 set was performed on GPL1352 [U133_X3P] Affymetrix Human X3P Array platform, GSE99394 set was per- Acridine orange assay for autophagy formed on [HTA-2_0] Affymetrix Human Transcriptome Array 2.0 Autophagy is characterized by formation of acidic vesicular [transcript (gene) version], and GSE56493 set was performed on organelles (AVO). To detect AVOs, acridine orange (AO) was [U133_X3P] Affymetrix Human X3P ArrayRosetta/Merck Human used. Acidic compartment causes accumulation of AO, which RSTA Custom Affymetrix 2.0 microarray [HuRSTA-2a520709] gives bright red fluorescence upon excitation by 488-nm laser. platform. Official gene symbols for the corresponding probes Measurement of red fluorescence is proportional to increase in were retrieved using DAVID (https://david.ncifcrf.gov/summary. AVOs (20). jsp) and expression array platform. Heat maps of combined Cells (2 105) were subjected to suspension culture for 48 datasets were generated using Morpheus (https://software.broad hours, after which they were trypsinized, counted, and 1 105 institute.org/morpheus/#). Semisupervised clustering was per- cells were stained with acridine orange (1 mg/mL) for 15 minutes formed to obtain function-based gene expression signatures. Box at 37C in DMEM þ 10% FBS. Post staining, cells were washed plots were plotted using GraphPad Prism 5.0 software. thrice in PBS. The cells were analyzed in BD FACS CantoII (Becton & Dickinson) equipped with a blue (488 nm) Coherent Sapphire Metastasis assay in mice Solid State laser. Red (564–606 nm) fluorescence emission from All animal experiments were reviewed and approved by the 104 cells was measured. Data were analyzed using Summit Institutional Animal Ethics committee of IISc, Bangalore. Cells V5.2.1.12465 software. (2 106) were resuspended in 50 mL DMEM and injected into the lateral tail vein of 5-week-old female NOD-SCID mice. Mice were Phosphatase activity assay sacrificed after 2 months of injection, and lungs and liver were For phosphatase activity assays, cell lysate was prepared in dissected to check for metastatic nodules. phosphatase activity buffer (20 mmol/L imidazole–HCl, pH 7.0, 2 mmol/L EDTA, 2 mmol/L EGTA, and protease inhibitor Statistical analysis cocktail). PP2A-Aa/b, PPM1E, and PP2C-a activities were mea- Statistical analyses were performed using GraphPad Prism 5.0 sured using Ser/Thr phosphatase assay kit (Cat. No. 17-127; software using Student t test. All data are presented as mean Millipore) according to the manufacturer's protocol. Briefly, SEM, where , P < 0.05; , P < 0.01; , P < 0.001.

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A B C D E Primary HMECs MDA-MB-231 MCF7 MDA-MB-231 MDA-MB-231

-70 -70 pAktS473 pAktT308 10.56 -70 Akt α-Tubulin -55 -70 pAMPKαT172 pPRAS40 -35 -70 T246 AMPKα 10.46

PRAS40 -35 α-Tubulin -55 α-Tubulin -55

14 10 pAMPK 14 pAMPK 5 12 pAMPK 12 * pACCS79 -250 8 * pAMPK 10 10 4 *** * 11.77 6 8 8 6 3 6 4 ACC 4 4 -250 2 2 2 2 α-Tubulin 1 1 1 1 -55 0.75 0.75 0.75 0.75 -colonies/Attached 0.5 AI-colonies/Attached AI 0.5 0.5 0.5 Suspension/Attached Relative expressionRelative * 0.25 Mammospheres/Attached 0.25 0.25 *** 0.25 pAkt ** *** pAkt pAkt pAkt

Figure 1. Matrix deprivation promotes Akt inactivation concomitant with AMPK activation. Representative immunoblots of the following cell lysates were probed for specified 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 for 7 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. Graphs represent densitometric quantification of immunoblots; error bars, mean SEM; n ¼ 3.

Results cultures. The levels of total Akt and total AMPKa proteins remained unchanged between these two conditions in all the Matrix deprivation leads to Akt inactivation concomitant to cell types. AMPK activation Consistent with mammospheres, subjecting MDA-MB-231 To begin to understand the interplay between Akt and AMPK cells to matrix detachment for 8 hours also resulted in a significant during the attachment–detachment cascade of metastasis, we first decrease in pAktS473 levels concomitant with increase in investigated their relative activities under these conditions in pAMPKaT172 levels (Fig. 1D). Further, phosphorylation of Akt breast cells. For this, we measured phosphorylation of Akt at at T308, which contributes to full Akt activation, was also reduced S473 (pAktS473) and phosphorylation of AMPKa at T172 in suspension (Fig. 1E). In keeping with their active phosphor- (pAMPKaT172) as surrogate measures of their activities (5, 21), ylation status, we also observed a decrease in the phosphorylation respectively. We recently reported AMPK activation in mammo- of PRAS40, an Akt substrate (22), and an increase in the phos- spheres formed by normal HMECs and spheroids formed by phorylation of ACC, an AMPK substrate (Fig. 1E; Supplementary breast cancer cells (16). Therefore, we first investigated the status Fig. S1B; ref. 23). We obtained similar results as early as 10 of Akt activity in these 3-dimensional spheroids. Interestingly, minutes of suspension culture (Supplementary Fig. S1B). In vitro concomitant with increase in the levels of pAMPKaT172, our study kinase assays further substantiated the reduction in Akt activity revealed a significant reduction in the levels of pAktS473 in and increase in AMPK activity in suspension (Supplementary Fig. anchorage-independent spheroids generated by HMECs (Fig. S1C). We also observed elevated pAMPKaT172 and reduced 1A), breast cancer cell lines MDA-MB-231 and MCF7 (Figs. 1B pAktS473 levels in several other matrix-deprived cancer cell lines and C), and primary breast cancer-derived cells (Supplementary of different epithelial origin (Supplementary Fig. S1D). Together, Fig. S1A), compared with their respective adherently growing these data revealed reciprocal regulation of AMPK and Akt

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activities between matrix-attached and matrix-detached condi- we investigated if AMPK might be directly involved in this process. tions, resulting in a pAkthigh/pAMPKlow state in adherently Interestingly, inhibition of AMPK using compound C led to growing cells, whereas a pAMPKhigh/pAktlow state in matrix- elevated pAktS473 levels in MDA-MB-231 (Fig. 3A), MCF7 (Sup- detached cells. plementary Fig. S3A), and BT474 (Supplementary Fig. S3B) cells. Further, overexpression of DN AMPK (Supplementary Fig. S3C) Akt repression in suspension is vital for anoikis resistance or depletion of AMPK using shRNA (Supplementary Fig. S3D) Intrigued by the reduction in Akt activity in detached breast also led to elevated pAktS473 levels in suspension, together reveal- cancer cells, contrary to its generally accepted role in anoikis ing a direct role for AMPK in Akt dephosphorylation in matrix- resistance, we examined the relative functional roles of Akt and deprived cells. AMPK in detached cells. Treatment of MDA-MB-231 cells with the Our observation that matrix deprivation led to Akt dephos- PI3K inhibitor LY294002 led to a decrease in pAktS473 levels phorylation in cells stably overexpressing myr-Akt, but not in cells (Supplementary Fig. S2A) while treatment with AMPK inhibitor stably overexpressing the double phosphomimetic Akt DD compound C (24) led to a decrease in pACCS79 levels (Supple- mutant (Figs. 2D and E), suggested the possible involvement of mentary Fig. S2B), confirming the efficacies of these pharmaco- phosphatases in this process. We, therefore, investigated the roles logic agents. Interestingly, inhibition of AMPK, but not Akt, of the Akt phosphatases PP2A and PHLPP. Inhibition of PP2A increased anoikis as revealed by elevated levels of cleaved cas- with okadaic acid failed to restore Akt phosphorylation in matrix- pase-3 as well as increase in caspase-3 enzymatic activity in detached cells (Supplementary Fig. S3E). Interestingly, we compound C treated cells (Fig. 2A and B). We obtained similar observed a significant increase in the protein levels (Fig. 3B) as results in MCF7 breast cancer cells also (Supplementary Fig. S2C). well as activity (Fig. 3C) of PHLPP2, which has specificity for Akt1 Consistent with these observations, we found that inhibition of (26), the major Akt isoform expressed by breast cancer cells (27), AMPK, but not Akt, abrogated anchorage-independent colony in matrix-deprived MDA-MB-231 cells. Further, knockdown of formation in MDA-MB-231, MCF7, and BT474 cells (Fig. 2C; PHLPP2 with two independent shRNA sequences led to signifi- Supplementary Fig. S2D and S2E). Interestingly, our observations cant increase in the levels of pAktS473 in suspension (Figs. 3D; in breast cancer cell lines also held true in HMEC-derived mammo- Supplementary Fig. S3F). In addition, shPHLPP2 cells also spheres, wherein again inhibition or knockdown of Akt failed to showed remarkable increase in the levels of pAktT308 in suspen- affect mammosphere formation (Supplementary Fig. S2F and S2G). sion (Supplementary Fig. S3G). These data suggested a role for Following this, we sought if it is necessary to maintain reduced PHLPP2 in Akt inactivation in suspension. Akt activity for stress survival in suspension. To address this, we Thereafter, we gauged if AMPK is involved in the observed evaluated the effects of forced Akt activation on anoikis by stably upregulation of PHLPP2. In the presence of AMPK inhibitor expressing a constitutively active HA myr-Akt construct in MDA- compound C (Fig. 3E), knockdown of AMPK (Fig. 3F) or over- MB-231 cells. Detection with antibodies against HA and total Akt expression of DN AMPK (Supplementary Fig. S3H), we observed confirmed exogenous protein expression (Fig. 2D). As expected, reduced levels of PHLPP2 in suspension in MDA-MB-231 cells. we observed increase in the levels of pAktS473 in these cells in Inhibition of AMPK in matrix-deprived MCF7 cells also led to a adherent condition (Fig. 2D). To our surprise, however, when decrease in PHLPP2 levels, in parallel with an increase in pAktS473 these cells were subjected to matrix deprivation, we failed to detect levels (Supplementary Fig. S3A). Also, we observed decreased elevated pAktS473 levels (Fig. 2D). We obtained similar results in levels of PHLPP2 upon overexpression of DN AMPK in adherent MCF7 cells transiently transfected with HA myr-Akt (Supplemen- MDA-MB-231 cells (Supplementary Fig. S3I) and in AMPKa / tary Fig. S2H), suggesting that myr-Akt is still susceptible to MEFs (Supplementary Fig. S3J). Consistent with these observa- negative regulation under matrix deprivation. tions, pharmacologic activation of AMPK with A-769662 (Sup- One major means of negative regulation of Akt is by dephos- plementary Fig. S3K), as well as genetic approach involving phorylation (5). To circumvent this, we used constitutively active, constitutively active CaMKK (Supplementary Fig. S3L), led to an GFP-tagged phosphomimetic HA-Akt-T308D S473D construct increase in the levels of PHLPP2 in adherent cells. These data thus (25), which is refractory to the action of phosphatases, and identified a novel positive regulation of PHLPP2 by AMPK. confirmed exogenous expression (Fig. 2E). Further, its expression We investigated potential mechanisms underlying AMPK- led to elevated Akt activity in adherent as well as matrix-deprived mediated upregulation of PHLPP2 in suspension. RT-PCR anal- cells, as gauged by increased levels of pPRAS40T246, a downstream yses revealed no significant change in the transcript levels of substrate of Akt (Fig. 2E). When these cells were subjected to PHLPP2 between matrix-attached and detached cells (both in the suspension for 48 hours, to our surprise, we observed increased presence and absence of AMPK inhibitor; Supplementary Fig. anoikis (Fig. 2F and G). Consistent with these data, overexpres- S4Ai). Further, we investigated the levels of miR-205, which has sion of GFP-HA-Akt-T308D S473D also impaired anchorage- been reported to regulate PHLPP2 levels (28). Quantitative PCR independent colony formation (Fig. 2H). We obtained similar analyses failed to detect significant change in the levels of miR-205 results in MCF7 cells transiently transfected with GFP-HA-Akt- between adherent and matrix-deprived conditions in MDA-MB- T308D S473D construct (Supplementary Fig. S2I and S2J), sug- 231 and MCF7 cells (Supplementary Fig. S4Aii). Further, inhibi- gesting that under the stress of matrix deprivation, hyperactiva- tion of AMPK also did not alter the levels of miR-205 in matrix- tion of Akt might be detrimental to cell survival. deprived cells (Supplementary Fig. S4Aii). Taken together, these observations were suggestive of possible posttranscriptional reg- AMPK-mediated stabilization of PHLPP2 promotes Akt ulation of PHLPP2 by AMPK. A cycloheximide chase assay inactivation in suspension revealed a more rapid decrease in the protein levels of PHLPP2 We next investigated the underlying mechanisms of Akt- in the presence of AMPK inhibitor (Fig. 3G). We obtained dephosphorylation in suspension. Because matrix deprivation similar data with cells expressing shAMPKa2 (Supplementary led to Akt dephosphorylation concomitant with AMPK activation, Fig. S4B), suggesting that AMPK might promote PHLPP2 protein

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B C A MDA-MB-231 2.5 *** * 120 ns DMSO LY CC 2.0 100 Cleaved -25 80 caspase-3 1.5 ns 60 a-Tubulin 1.0 -55 40 0.5

random fields random 20 # of colonies in 15 # of colonies Caspase-3 activity 0.0 0

D MDA-MB-231 E MDA-MB-231, Att MDA-MB-231, Sus Att Sus GFP +- +- Ctrl V +- +- GFP Akt DD -+ -+

HA myrAkt -+ -+ T246 pPRAS40 -35 -70 -35 pAktS473 15.89 16.17

1 1.46 0.08 0.05 PRAS40 -35 -70 -35 Akt Akt -70 -70 -70 HA a-Tubulin -55 -55 a-Tubulin -55

F G H MDA-MB-231, Sus * 150 2.5 *** GFP +- 2.0 GFP Akt DD -+ 100 1.5 Cleaved -25 caspase-3 1.0 50

0.5 random fields random a-Tubulin # of colonies in 15 # of colonies -55 Caspase-3 activity 0.0 0 GFP GFP Akt DD GFP GFP Akt DD

Figure 2. 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 of suspension and harvested for immunoblotting (A) and caspase-3 activity assay (B); n ¼ 3. Treated cells were subjected to colony formation in methylcellulose for 15 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 for cleaved 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 two experiments with three dishes each.

stabilization. However, MG132 failed to restore protein levels of Fig. S4E), suggesting that AMPK might mediate PHLPP2 stability PHLPP2 under AMPK inhibited condition (Supplementary Fig. in detached cells by regulating lysosomal degradation. S4C). Also, ubiquitination of PHLPP2 did not change between attached and matrix-deprived cells (Supplementary Fig. S4D), PHLPP2 knockdown inhibits autophagy and promotes anoikis suggesting proteasome-independent mechanism of PHLPP2 sta- To further understand the biological signiﬁcance of AMPK- bilization. Interestingly, lysosomal inhibitors restored PHLPP2 mediated Akt inactivation through PHLPP2, we investigated the protein levels under AMPK inhibited condition (Supplementary effects of PHLPP2 knockdown in anoikis and anchorage-

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A MDA-MB-231, Sus B MDA-MB-231 C. MDA-MB-231 Sus PHLPP2 activity DMSO CC 0.8 Att 10 min 8 hrs *** -70 pAktS473 0.6 1 1.89 PHLPP2 -130 0.4 -70 Akt 1 2.40 2.62 0.2 a-Tubulin -55 a-Tubulin -55

Absorbance at 640 nm 640 at Absorbance 0.0

IgG Att Sus

D MDA-MB-231, Sus E MDA-MB-231, Sus F MDA-MB-231, Sus

shPHLPP2 DMSO CC shAMPKα2 NT (seq #5) EV (seq #1) PHLPP2 -70 -130 pAktS473 10.40 PHLPP2 -130 1 10.9 a-Tubulin 10.51 -55 -70 Akt -70 AMPKα2 10.02 PHLPP2 -55 -130 a-Tubulin 10.37 a-Tubulin -55

G MDA-MB-231, Sus DMSO CC CHX: Time (hrs) 0 4 86 046 8

PHLPP2 -130

a-Tubulin -55

1.5 DMSO CC 1.0

0.5 PHLPP2 Levels (relative to time 0) 0.0 0246810 Time (hrs) of CHX treatment in Sus

Figure 3. 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 immunoblots of MDA-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 stably expressing pTRIPZ empty vector (EV) or shAMPKa2 (seq #1) were induced with doxycycline for 48 hours, followed by suspension for 8 hours; n ¼ 3. G, Adherent cells pretreated 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.

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independent colony formation. When subjected to suspension for and anchorage-independent growth were partially reversed in the 48 hours, we observed increased levels of cleaved caspase-3 presence of LY294002 (Fig. 4A and B; Supplementary Fig. S5A), (Supplementary Fig. S5A) as well as elevated caspase-3 enzyme suggesting that PHLPP2 depletion promotes anoikis and impairs activity (Fig. 4A) in shPHLPP2 (seq #5) cells as compared with anchorage-independent growth, at least in part, through Akt control cells expressing nontargeting (NT) shRNA. Similar obser- activation. vations were made with an independent shPHLPP2 (seq #3) Recent studies have revealed the induction of autophagy as a expressing cells (Supplementary Fig. S5B) as well as in MCF7 critical survival strategy for anoikis resistance (29). Because Akt cells (Supplementary Figs. S5C and S5D). Consistent with this, inhibits autophagy (30) and PHLPP2 knockdown cells showed PHLPP2 knockdown cells formed signiﬁcantly reduced number Akt activation, we investigated if impairment of autophagy is of colonies in methylcellulose (Figs. 4B; Supplementary Fig. S5E responsible for anoikis in these cells. Compared with control and S5F). Moreover, the effects of PHLPP2 depletion on anoikis NT cells, we observed reduced AO-Red ﬂuorescence (Fig. 4C) and

A B C D 150 5 ** ** 800 * 250 ** ity 4 *** 200 in 15 100 600 3 150 -3 activ

e 400

s 2 50 100 200 AO-Red (MFI) AO-Red (MFI) 1 of colonies 50 random fields Caspa #

0 0 0 0 LY - + LY - - + LY - - + NT shPHLPP2 (seq #5) shPHLPP2 NT shPHLPP2 NT shPHLPP2 (seq #5) (seq #5) (seq #5) E F 800 MDA-MB-231, Sus 600 * GFP +- GFP Akt DD -+ 400 LC3B-I LC3B-II -15 200 AO-Red (MFI) α-Tubulin -55 0 GFP GFP Akt DD H G Lungs Liver Lungs Liver 10 10 10 *** * 10 *** 8 8 * 8 8

nodules 6

nodules 6 nodules

6 nodules 6

4 4 4 4 sible sible i sible i sible i v i v

v 2 2 2 v 2 # of

0 # of 0

# of 0 0 # of NT shPHLPP2 NT shPHLPP2 GFP GFP Akt DD GFP GFP Akt DD (seq #5) (seq #5)

Figure 4. Downregulation of PHLPP2 promotes anoikis and impairs autophagy and metastasis. A–D, MDA-MB-231 cells stably expressing nontargeting shRNA (NT) or shPHLPP2 (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), subjected to 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-T308D S473D (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, Graphs represent number of lung (left) and liver (right) nodules following tail-vein injection of cells described below; each dot represents number of nodules in a single 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.

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decreased levels of LC3B-II (Supplementary Fig. S5A) in MDA- pAMPKaT172 levels in adherent cells (Fig. 5D). Further, inhibition MB-231 shPHLPP2 cells. We obtained similar results in MCF7 of Akt, while it did not cause a change in PP2C-a activity cells transiently transfected with shPHLPP2 construct (Supple- (Supplementary Fig. S6H), impaired the interaction between mentary Fig. S5G), indicating reduced autophagy upon PHLPP2 AMPK and PP2C-a in adherent cells (Fig. 5E), together suggesting depletion. Furthermore, treatment with LY294002 restored an Akt-dependent, PP2C-a–mediated dephosphorylation of autophagy induction in matrix-deprived shPHLPP2 cells (Fig. AMPK in adhesion. 4D; Supplementary Fig. S5A), suggesting that elevated Akt activity Thus, our data showed that while matrix detachment-triggered in these cells might be one possible reason for reduced autophagy. AMPK activation inhibits Akt, attachment-triggered Akt activation Consistent with this notion, we observed that MDA-MB-231 cells inhibits AMPK. These data are suggestive of a double-negative stably expressing GFP-HA-Akt-T308D S473D also displayed feedback loop between these two kinases in matrix-adhered reduced AO-Red fluorescence and decreased levels of LC3B-II, versus matrix-detached states of cells. To further confirm this, we indicating reduced autophagy (Fig. 4E and F). Thus, our observa- tested the effect of forced activation of AMPK on pAktS473 levels in tions suggest that AMPK-mediated suppression of Akt activity adherent conditions (where normally AMPK activity is low) and possibly facilitates anoikis resistance through facilitating the forced activation of Akt on pAMPKaT172 levels in suspension induction of autophagy. (where normally Akt activity is low). Activation of AMPK in Because anoikis resistance contributes to metastatic potential adherent conditions, mediated by AMPK activator A-769662 (Fig. of cancer cells, we investigated the effects of PHLPP2 knock- 5F; Supplementary Fig. S7A), overexpression of constitutively down or overexpression of phosphomimetic Akt on metastasis. active AMPK-upstream kinase CaMKK (Fig. 5G; Supplementary Tail-vein injections in nude mice revealed that MDA-MB-231 Fig. S7B), or knockdown of PP2C-a (Fig. 5H; Supplementary Fig. cells stably expressing shPHLPP2 (Fig. 4G; Supplementary Fig. S7C), promoted Akt inactivation. On the other hand, forced S5H) or Akt-T308D S473D (Fig. 4H; Supplementary Fig. S5I) activation of Akt in suspension, as observed in cells stably expres- were impaired in their metastatic potential, thus corroborating sing phosphomimetic Akt-T308D S473D (Fig. 2E) or shPHLPP2 our observations of increased anoikis and decreased anchorage- (Fig. 3D), promoted AMPK inactivation (Fig. 5I and J; Supple- independent growth. mentary Fig. S7D and S7E). Collectively, these data identify a novel double-negative feedback loop between the two cellular Adhesion-dependent double-negative feedback loop between kinases Akt and AMPK, which maintains an attachment-triggered AMPK and Akt pAkthigh/pAMPKlow state in adherent cells while maintaining Our data thus far revealed that detachment-triggered AMPK a detachment-triggered pAMPKhigh/pAktlow state in matrix- leads to Akt inactivation, which is critical for stress survival under deprived cells. detachment. We reasoned that after exiting the circulation and To further corroborate the double-negative cross-talk between following attachment at the new site, proliferative signals are AMPK and Akt a microarray-based gene expression analysis was critical for secondary tumor growth, which might require reacti- performed between adherent and detached MDA-MB-231 cells. vation of Akt and inactivation of AMPK. Therefore, we investi- Consistent with higher levels of pAkt in adherent cells, we gated the status of AMPK and Akt signaling in cells subjected to observed elevated Akt gene signature, including upregulation of reattachment following matrix detachment. Reattachment of AKT1, CDC34, NEDD8, PRKCD, and DUSP10 (31) in adherent MDA-MB-231 (Fig. 5A) and MCF7 (Supplementary Fig. S6A) condition. Further, in keeping with a role for Akt signaling in cells following matrix deprivation quickly led to restoration of anabolic pathways, we observed elevated expression of genes pAktS473 levels comparable with originally adherent cells. Con- involved in fatty acid synthesis (such as ACAA1 and 2, ACAD comitant with reattachment, we observed rapid dephosphoryla- 10 and 11, ACOX 1 and 2, ACSBG 1 and 2, and ACSL 3, 5, and 6) tion of AMPK, as early as 1 hour of reattachment, in both MDA- and pentose phosphate pathway (such as G6PD, H6PD, PGLS, MB-231 (Fig. 5A) and MCF7 (Supplementary Fig. S6A) cells. PRPS1, and PRPS1L1; ref. 32). Additionally, we observed elevated Further, reattachment of cells in the presence of Akt inhibitor expression of genes involved in the mTOR pathway and protein impaired the attachment-triggered dephosphorylation of AMPK synthesis (such as EIF4B, HIF1A, RPS6, EIF4EBP1, EIF4EBP2, in both MDA-MB-231 (Fig. 5B) and MCF7 (Supplementary Fig. PPP2CA, RPS6, and IKBKB; ref. 31) and in cell growth and S6B) cells, suggesting a direct role for Akt in AMPK dephosphor- proliferation (such as ANAPC2, CDK4, CDK6, CDKN3, CUL1, ylation following adhesion. E2F1, SKP2, CDC6, and WEE1; ref. 33) in adherent cells (Fig. 6A We sought to investigate the role of phosphatases in the and B). adhesion-mediated dephosphorylation of AMPK. We did not Similarly, AMPK activation in matrix-detached cells was detect any differences in the levels or activities of PP2A-Aa/b, supported by the upregulation of AMPK gene signature, includ- PPM1E, and the monomeric phosphatase PP2C-a, that have been ing PTPLB, SFXN1, FKBP5, TUSC5, ORM1, CEBP1, MAP3K6, identified as cellular AMPK phosphatases, between adhered and and PRKAR2B (Fig.6A;refs.18and34).Additionally,genes detached conditions (Supplementary Fig. S6C and S6D). In involved in catabolic processes including fatty acid oxidation immunoprecipitation experiments using individual phospha- (such as HSD17B4, ACOT8, ACOX3, ABCD1,andACOX1; tase-specific antibodies, we observed no change in the interaction ref. 32), and glycolysis (such as ALDOA, ALDOC, ENO1,2,3, between AMPK and PP2A-Aa/b or PPM1E between adherent and GALM, HK2, G6PD,andALDOB; ref. 32), and genes involved in detached cells (Supplementary Fig. S6E and S6F). Interestingly, stress-responsive pathways including autophagy, oxidative we observed a noticeable increase in the interaction between stress, and hypoxia (such as ATG4, ATG9, GABARAPL1, IRGM, AMPK and PP2C-a under adhesion compared with detachment MAP1LC3A,andULK1; ref. 35) were elevated in detached (Fig. 5C). A reverse pulldown using AMPKa2-specific antibodies MDA-MB-231 cells (Fig. 6C). further confirmed these results (Supplementary Fig. S6G). Moreover, AMPK knockdown reversed the expression of several Consistent with this, PP2C-a knockdown led to increased of these genes in matrix-detached cells (Fig. 6A–C), revealing their

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MCF7 A MDA-MB-231 B MDA-MB-231 C Re-att, 1 hr IP: PP2C-α DMSO Akti VIII

-70 pAMPKαT172 -70 S473 pAkt -70 -70 AMPKα AMPKα2 -70 Akt α a-Tubulin -55 PP2C- -35 -70 pAMPKαT172 -70 Input pAktS473 -70 α AMPK -70 Akt a-Tubulin -55 α PP2C- -35 a -Tubulin -55 a-Tubulin -55

DEMCF7 MCF7 MCF7 IP: PP2C-α

-70 -70 pAMPKαT172 pAktS473 -70 AMPKα2 PP2C-α -35 PP2C-α -35 α a -55 PP2C- -Tubulin a -55 -Tubulin -55 FIGH J Att Att Att Sus Sus 1.5 1.5 1.5 1.5

1.5 levels levels levels levels levels T172 S79 *** * α *** S473 S473 S473 * 1.0 1.0 1.0 1.0 1.0

0.5 0.5 0.5 0.5 0.5 Relative pACC Relative Relative pAkt Relative pAkt Relative Relative pAkt Relative Relative pAMPK Relative 0.0 0.0 0.0 0.0 0.0

Figure 5. 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 to 10 minutes of suspension (Sus) were allowed to reattach (Re-att) in the presence of DMSO or Akt inhibitor; n ¼ 3. C, Lysates of MCF7 cells cultured in conditions of attachment (Att), suspension (Sus), and reattachment (Re-att) were immunoprecipitated (IP) with control IgG or anti–PP2C-a antibodies and analyzed by immunoblotting. The input represents 2% of the whole-cell lysate used for each immunoprecipitation; n ¼ 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 described in C; n ¼ 3. F–J, Graphs represent densitometric quantiﬁcation of immunoblots (Supplementary Figs. S7A-S7E) for relative phospho-protein levels from cells harvested 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 Supplementary Fig. S7B); n ¼ 3. H, Adherent MCF7 cells transfected with vector control (pLKO.1) or shPP2C-a (also see Supplementary Fig. S7C); n ¼ 2. I, MCF7 cells transfected with GFP 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 expressing nontargeting shRNA (NT) or shPHLPP2 (seq #5) and subjected to suspension (48 hours; also see Supplementary Fig. S7E); n ¼ 3. Error bars, mean SEM.

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A B Cell growth Akt pathway AMPK pathway and proliferation Cellular stress AKT1 PTPLB ANAPC2 CDC34 AMBRA1 SFXN1 CCND1 MVK ATG16L1 FKBP5 CCNE1 PTEN NEDD8 ATG4B TUSC5 SLC36A2 CDK4 TCL1A PRKCD ATG4D ORM1 ACP5 CDK6 HSPB1 UBE2M ATG9A RETN HP CDKN3 ILK DUSP10 ATG9B CIDEC CYP2E1 CUL1 BTK PMPCA GABARAPL1 PIK3CD MCF2L CUL2 PDK2 ADIPOR1 GABARAPL2 FABP4 AOC3 E2F1 GPX4 KCTD5 IRGM ADRB3 CFD SKP2 LASP1 TOLLIP MAP1LC3A IER5 ADIPOQ CDC6 MMP15 RNF126 ULK1 LEP AACS WEE1 CLSTN1 DKK1 CDKN1B WIPI1 CEBPA SLC7A8 ANAPC2 BRMS1 JAG1 CUL3 DRAM1 Autophagy genes AGPAT2 SLC25A1 CDC25A TJP3 Jun ABL1 LAMP1 FASN PPARG CDK5R1 FGF2 BIK SERTAD1 NPC1 BNIP3 MGST1 ARRDC2 CDK7 IGFBP2 BMPER CDC25C CYP1A1 TKT DGAT2 CDKN3 ATG12 Oxidative BMP4 NOS3 STMN1 GPX3 PYGL FMO2 CKS1B Ang ANGPT2 ATM ATG4A PRDX2 ELOVL6 CKS2 Stress IGFBP3 CHDH ATG4C BMP2 MAP3K6 CDKN2B ATM IL6 SLC7A10 GTSE1 CDH2 CIDEA RBL2 ATG5 GADD45G DLL1 PPP1R3C KPNA2 DNA damage FST ACLY TP53 GABARAP TP53 DLL4 ITGB1BP3 MNAT1 CXCL12 THRSP MAP1LC3B XPC JAG2 FOXO1 EIF4B AURKB Hypoxia FSTL3 LIPE CSTB PCK1 EIF4E CCNB2 RGS19 Csf2 AKT2 RARA Signaling PRKAR2B UCP1 CCNF GABARAP Traf1 AKT3 EIF4EBP1 DNAJC15 SLC1A5 CDO1 CDC6 Heat shock Trib3 GRB10 EIF4EBP2 PRDX2 CDC16 Cd69 GRB2 ADAMTSL4 PPP2CA COX2 Protein CDC20 Cd52 MTCP1 ANKRD2 PPP2R2B SCARA3 MRE11A Gata3 PDPK1 TIMP4 PPP2R4 BRCA1 RAD51 Egr1 PIK3CG XDH RPS6 CCND1 BRCA1 PIK3R1 NNAT Translation RPS6KA1 GADD45A CASP3 CSRP3 RPS6KA2 MLH1

PAK1 mTOR-pathway/ RBL1 TMEM45B RPS6KA5 MSH2 LMOD2 RPS6KB1 INSIG1 MYL2 RPS6KB2 UBE2G2 SYP

C D E

Fatty acid Fatty acid synthesis Att Sus shAMPK, Sus Att Sus shAMPK, Sus b-oxidation CDH3 ACAA1 ACAT1 CDH4 Cell-Cell HSD17B4 ACAA2 ACAT2 CDH5 ACAD9 ACOT8 CD44 Adhesion ACAD10 ACADL ACOX3 ITGA1 ACAD11 ABCD1 ACADM ALDOA ITGA2 ACADS ACADSB ACOX1 ALDOC ITGA3 CRAT ACADVL GCDH ENO1 ITGA5 ACOX3 ACOX2 EHHADH ENO2 ITGA6 ACSL1 ACAA1 ENO3 ITGA7 ACOX1 ACSL4 EHHADH GALM ITGA8 Cell-extracellular ACOX2 ACSM3 SCP2 HK2 ITGAL ACSBG1 ACSM5 ACOXL PFKL ITGAM matrix (ECM) ACOT6 Glycolysis ACSBG2 ECI2 PGAM2 ITGAV adhesion ACOT9 AMACR PGK1 ITGB1 ACSL3 CROT CROT PGK2 ITGB3 ACSL5 DECR2 PGM1 ITGB4 ACSL6 ECHS1 PKLR ITGB5 G6PD ECI2 TPI1 SGCE H6PD PECR G6PD SPP1 Primary CTC Metastasis PGLS PPA1 H6PD PRPS1 PGLS PRPS1L1 RBKS Z-score PPP PRPS2 ALDOB RBKS BPGM RPE GCK RPIA 6 TALDO1 TKT F

4 Primary CTC 2 Metastasis

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-2

-4 ACLY RETN NPC1 MAP1LC3B CYP1A1 BRCA1 UBE2G2 NNAT ANKRD2 LIPE

Figure 6. Microarray-based expression proﬁling of genes regulated in matrix-attached versus matrix-detached states of cells. A–D, Heat map depicting unsupervised clustering 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 Akt and 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 show distribution of normalized (Z-score) gene expression of AMPK-dependent genes from microarray data available for primary tumor (GSE43837), CTCs (GSE99394), and metastases of breast cancer patients (GSE43837 and GSE56493).

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MATRIX-ATTACHED MATRIX-DEPRIVED Figure 7. high low pAkt /pAMPK pAMPKhigh/pAktlow A model for double-negative feedback Detachment loop between two cellular kinases AMPK and Akt. We show that detachment-triggered AMPK AMPK AMPK concomitantly inactivates Akt through the phosphatase PHLPP2, resulting in a pAMPKhigh/pAktlow catabolic state PP2C-α PHLPP2 that facilitates stress-survival in matrix-deprived cells. In contrast, adhesion-triggered Akt keeps AMPK Akt Akt under check by the phosphatase PP2C-a, maintaining a pAkthigh/ pAMPKlow anabolic state, which is conducive for cell growth and Attachment proliferation. Thus, between Akt/mTOR pathway AMPK pathway attachment and detachment, AMPK Anabolic state Catabolic state and Akt constitute a reversible Fatty acid biosynthesis Fatty acid oxidation double-negative feedback loop, Protein translation Glycolysis maintaining stable pAkthigh/pAMPKlow high low Cell cycle Autophagy and pAMPK /pAkt states, yet retaining the ability to switch between Cell growth and proliferation Stress-survival these two cellular states.

AMPK dependency. We also observed AMPK-dependent altered deprivation during transit through the circulation. Yet, what expression of integrins that are known to be involved in anoikis maintains the states of adhesion, characterized by cell growth resistance and contribute to metastasis (36) in detached cells (Fig. and proliferation, versus the state of matrix-detachment, charac- 6D). In addition, microarray data analyses of matrix-detached terized by stress survival, remains poorly understood. In this PHLPP2-knockdown cells and GFP-HA-Akt-T308D S473D- study, we show that AMPK activation triggered by matrix depri- expressing cells also exhibited upregulation of the Akt pathway vation leads to a concomitant inactivation of Akt by stabilizing and apoptotic genes while showing downregulation of autop- PHLPP2 in breast cancer cells. Knockdown of PHLPP2 or consti- hagy-related genes (Supplementary Fig. S7F–S7H), thus support- tutive activation of Akt increased anoikis while impairing autop- ing our experimental data. Collectively, these data supported the hagy, thus inhibiting anchorage-independent growth and metas- concept of double-negative feedback loop between Akt and AMPK tasis, and highlighting the importance of suppression of Akt in matrix-adhered versus detached states of cells. activity for surviving the stress of matrix deprivation. We further In order to understand the functional relevance of the double- demonstrate that matrix reattachment-triggered Akt activation negative feedback between Akt and AMPK in breast cancer pro- concomitantly promotes AMPK inactivation through yet another gression, we examined publicly available microarray data from phosphatase PP2C-a. Our data, thus, reveal for the ﬁrst time a the GEO of patient-derived primary breast tumors (GSE43837), reversible, double-negative feedback loop between AMPK and Akt circulating tumor cells (GSE99394), and metastatic lesions at between matrix-adhered versus detached states that can coordi- different organs (GSE43837 and GSE56493). Similar to adher- nate the intracellular signaling pathways involved in cell growth ently growing MDA-MB-231 cells (Fig. 6 A–D), heat map gener- and stress survival during cancer progression. ated from cDNA microarrays of patient samples revealed an Akt Akt signaling represents a widely accepted prosurvival pathway pathway associated gene expression pattern in primary and met- that is known to aid anoikis resistance and tumor progression astatic lesions (Fig. 6E) that was suppressed in CTCs. In contrast, (38). Surprisingly, we observed Akt repression concomitant with and similar to detached MDA-MB-231 cells (Fig. 6B), we observed AMPK activation in a large array of matrix-deprived epithelial AMPK-dependent gene expression, including those of stress sig- cancer cells (of lung, glial, cervix, liver origin) including breast naling and autophagy, in patient-derived CTC microarray data cancer cell lines MDA-MB-231 and MCF7. This prompted us to that were suppressed in primary and metastatic lesions (Fig. 6F). investigate if suppression of Akt activity in suspension is required For example, box plot analysis showed AMPK-dependent upre- for cell survival. To address this, we enforced Akt activation in gulation of ACLY, RETN (18), and autophagy-related genes NPC1, matrix-deprived cells using constitutively active Akt constructs. LC3B, CYP1A1, BRCA1, and UBE2G2 (37), while revealing down- Interestingly, even though both myr-Akt and the phosphomi- regulation of NNAT, ANKRD2, and LIPE (Fig. 6F; ref. 18) in CTCs. metic mutant Akt-T308D S473D led to elevated Akt activity in adherent cells, we detected increased Akt activity in suspension only with the expression of Akt-T308D S473D, but not with myr- Discussion Akt, an oft-used construct throughout literature. Based on our Once tumors metastasize to a distal site, they are mostly fatal to ﬁndings that matrix deprivation upregulates the Akt phosphatase the patient due to lack of strategies currently to treat metastasis. PHLPP2, we predict that myristoylated, membrane-tethered Therefore, understanding the molecular mechanisms that con- forms of Akt are still susceptible to negative regulation in sus- tribute to cancer metastasis can advance treatment approach. pension by phosphatases. Interestingly, expression of Akt-T308D Tumor cells grow adherently both in primary and secondary S473D, which is refractory to dephosphorylation at the key tumor sites, but they need to overcome the stress of matrix activating phosphorylation sites, promoted anoikis, suggesting

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that high Akt activity might be detrimental to cancer cell survival activity leading to AMPK inactivation through increased interac- in suspension. This is in agreement with yet another report that tion between AMPK and its phosphatase, PP2C-a. While PP2C-a showed elevated Akt activation leads to cell death due to increase is predominantly nuclear localized (47), AMPK is reported to in reactive oxygen species (39). show nuclear localization and cytoplasmic redistribution through Our data revealed AMPK-dependent upregulation of PHLPP2 Ran-dependent import and CRM1-mediated export pathways in suspension. However, we failed to detect changes in transcript (48). Interestingly, Akt has been shown to regulate RanBP and levels of PHLPP2, suggesting possible posttranscriptional regula- CRM1-dependent nuclear-cytoplasmic shuttling of proteins (49). tion. Although PHLPP2 has been shown to be targeted for Thus, one possible way by which Akt could regulate the interac- ubiquitin-mediated degradation (40), we failed to see changes tion between AMPK and PP2C-a might involve Akt-dependent in its ubiquitination or effect of inhibition of proteasomal deg- redistribution of AMPK. radation. Instead, our data suggested a possible role for lysosomal These findings, thus, identify a novel, and reversible, double- degradation in regulating PHLPP2 protein levels. Interestingly, a negative feedback loop between AMPK and Akt between matrix- recent paper has demonstrated a role for PHLPP1 and Akt in the attached and matrix-deprived conditions. Comparison of micro- regulation of chaperone-mediated autophagy that regulates pro- array gene expression data of primary tumors, circulating tumor tein degradation (41). However, a selective chaperone-dependent cells, and metastatic lesions further suggested that such a recip- targeting of PHLPP2 for lysosomal degradation remains to be rocal regulation might exist in metastatic breast cancers. We explored. Yet another possible mechanism of PHLPP2 upregula- propose that such a reversible regulation might help rapid switch- tion could involve rapid protein synthesis as we observed elevated ing between pAkthigh/pAMPKlow state in adhesion that favors cell PHLPP2 levels within 10 minutes of suspension. Because AMPK growth, to pAMPKhigh/pAktlow state that allows adaptation to activation is known to inhibit cap-dependent translation in matrix-detachment stress (Fig. 7), yet retaining the ability to restore suspension (18), this might possibly involve stress-associated growth following proper attachment at the secondary site. Disrupt- alternate modes of translation. ing this loop using AMPK or PHLPP2 inhibitors might provide Reduced PHLPP2 protein expression in colon cancer and novel therapeutic strategies to restrict metastatic cancer spread. pancreatic ductal adenocarcinoma patient samples, and anti- tumorigenic effects of its overexpression in colon and pancreatic Disclosure of Potential Conflicts of Interest cancer cell lines (42, 43) have largely supported tumor-suppres- No potential conflicts of interest were disclosed. sive functions for PHLPP2. In contrast, our results showed that depletion of PHLPP2 rendered breast cancer cells anoikis-sensi- Authors' Contributions tive and impaired metastasis. In support of this, a recent study Conception and design: M. Saha, S. Kumar, S.K. Hindupur, A. Rangarajan using PTEN/TP53-mutant prostate cancer mice showed that myc Development of methodology: M. Saha, S. Kumar, S. Bukhari, S.A. Balaji, S.K. Hindupur drives proliferation and metastasis through activation of PHLPP2 fi Acquisition of data (provided animals, acquired and managed patients, and suppression of the Akt pathway (44). Together, these ndings provided facilities, etc.): M. Saha, S. Kumar, S.A. Balaji begin to highlight novel, context-specific tumorigenic functions Analysis and interpretation of data (e.g., statistical analysis, biostatistics, for PHLPP2. computational analysis): M. Saha, S. Kumar, S. Bukhari, P. Kumar, We report here an AMPK-mediated suppression of Akt activity S.K. Hindupur, A. Rangarajan in matrix-deprived cell survival. We speculate that such a negative Writing, review, and/or revision of the manuscript: M. Saha, S. Kumar, S. Bukhari, P. Kumar, S.K. Hindupur, A. Rangarajan regulation of Akt by AMPK might be favorable to matrix-deprived Administrative, technical, or material support (i.e., reporting or organizing cells because this can shift cellular signaling from energy-con- data, constructing databases): M. Saha, A. Rangarajan suming/anabolic processes (mediated by Akt activation) to ener- Study supervision: A. Rangarajan gy producing/catabolic processes (mediated by AMPK activation), thus restoring energy homeostasis. This is consistent with a recent Acknowledgments finding revealing AMPK-mediated inhibition of mTOR and sup- This work was supported by the Wellcome Trust/DBT India Alliance Fel- pression of protein synthesis in anoikis resistance (18). Further, lowship (grant number 500112-Z-09-Z) awarded to A. Rangarajan. Grants from the catabolic process of autophagy, which is also known to DBT-IISc partnership program and support from DST-FIST and UGC, Govern- ment of India, to the Department of MRDG are also acknowledged. regulate energy balance, plays a key role in anoikis-resistance The authors thank Drs. M. Vijaya Kumar and Rekha V. Kumar for their help in (29). Interestingly, Akt activation has been shown to inhibit procuring primary tissue samples at KMIO; Drs Benoit Viollet for AMPK DKO autophagy (30). In keeping with this, our present data showed cells and Deepak Saini for shRNA of phosphatases; Sukrutha Reddy, Nehanjali that PHLPP2 knockdown or enforced Akt activation in matrix- Dwivedi, and Sunaina Rao for help with immunoblotting; and Srikanth S. deprived cells inhibited autophagy and increased anoikis. Thus, Manda, Institute of Bioinformatics, for analysis of microarray data. The authors our data suggest that AMPK-mediated Akt inactivation might acknowledge the Central Animal Facility and FACS facility at IISc. additionally contribute to anoikis resistance by promoting The costs of publication of this article were defrayed in part by the payment autophagy. of page charges. This article must therefore be hereby marked advertisement Matrix attachment leads to Akt activation through integrin in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. signaling (45), while we have identified elevated calcium and ROS levels as triggers for detachment-induced AMPK activation Received July 17, 2017; revised November 17, 2017; accepted January 10, (46). We show here that reattachment to the matrix restores Akt 2018; published OnlineFirst January 16, 2018.

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1510 Cancer Res; 78(6) March 15, 2018 Cancer Research

AMPK−Akt Double-Negative Feedback Loop in Breast Cancer Cells Regulates Their Adaptation to Matrix Deprivation

Manipa Saha, Saurav Kumar, Shoiab Bukhari, et al.

Cancer Res 2018;78:1497-1510. Published OnlineFirst January 16, 2018.

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