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. Ó2018 AACR. Introduction vival, and metabolism, and plays a major role in tumor progres- sion (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 Ó2018 American Association for Cancer Research. breast cancer cells sensitive to anoikis (15). Thus, the role of Akt in www.aacrjournals.org 1497 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst January 16, 2018; DOI: 10.1158/0008-5472.CAN-17-2090 Saha et al. 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