Published OnlineFirst February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2215 Cancer Metabolism and Chemical Biology Research RSK Regulates PFK-2 Activity to Promote Metabolic Rewiring in Melanoma Thibault Houles1, Simon-Pierre Gravel2,Genevieve Lavoie1, Sejeong Shin3, Mathilde Savall1, Antoine Meant 1, Benoit Grondin1, Louis Gaboury1,4, Sang-Oh Yoon3, Julie St-Pierre2, and Philippe P. Roux1,4 Abstract Metabolic reprogramming is a hallmark of cancer that includes glycolytic flux in melanoma cells, suggesting an important role for increased glucose uptake and accelerated aerobic glycolysis. This RSK in BRAF-mediated metabolic rewiring. Consistent with this, phenotypeisrequiredtofulfill anabolic demands associated with expression of a phosphorylation-deficient mutant of PFKFB2 aberrant cell proliferation and is often mediated by oncogenic decreased aerobic glycolysis and reduced the growth of melanoma drivers such as activated BRAF. In this study, we show that the in mice. Together, these results indicate that RSK-mediated phos- MAPK-activated p90 ribosomal S6 kinase (RSK) is necessary to phorylation of PFKFB2 plays a key role in the metabolism and maintain glycolytic metabolism in BRAF-mutated melanoma growth of BRAF-mutated melanomas. cells. RSK directly phosphorylated the regulatory domain of Significance: RSK promotes glycolytic metabolism and the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2), growth of BRAF-mutated melanoma by driving phosphory- an enzyme that catalyzes the synthesis of fructose-2,6-bisphosphate lation of an important glycolytic enzyme. Cancer Res; 78(9); during glycolysis. Inhibition of RSK reduced PFKFB2 activity and 2191–204. Ó2018 AACR. Introduction but recently developed therapies that target components of the MAPK pathway have demonstrated survival advantage in pati- Melanoma is the most aggressive form of skin cancer and arises ents with BRAF-mutated tumors (7). Drugs that inhibit mutated from melanocytes, which reside in the skin, eye, mucosal epithe- BRAF (vemurafenib, dabrafenib) lead to tumor regression and lia, and meninges of the brain and spinal cord (1). In cutaneous improved overall patient survival (8–10). Despite the promise of melanoma, the RAS/MAPK pathway is frequently activated due BRAF inhibitors in the clinic, intrinsic and acquired resistances to mutations in BRAF (52%), NRAS (28%), and NF1 (14%) were found to limit the therapeutic benefit of these drugs (11). genes (2). More than 90% of BRAF mutations encode a protein The RAS/MAPK signaling cascade regulates a wide range of harboring the V600E mutation, which constitutively activates ERK cellular functions, including cell growth, proliferation, survival, 1/2 signaling and drives cell proliferation (3–5). Malignant mel- and metabolism (12, 13). Upon activation by upstream RAS anoma is highly resistant to conventional chemotherapy (6), isoforms or in response to mutational activation, RAF phosphory- lates and activates MEK1/2, which themselves phosphorylate and activate ERK1/2 (14). Once activated, ERK1/2 phosphorylate 1Institute for Research in Immunology and Cancer (IRIC), Universite de Montreal, many substrates, including members of the p90 ribosomal S6 2 Montreal, Quebec, Canada. Department of Biochemistry, Goodman Cancer kinase (RSK) family of proteins (15). The RSK family comprises of Research Centre, McGill University, Montreal, Quebec, Canada. 3Department of four Ser/Thr kinases (RSK1–4) that share 73% to 80% sequence Cancer Biology & Pharmacology, University of Illinois College of Medicine, Peoria, Illinois. 4Department of Pathology and Cell Biology, Faculty of Medicine, identity and belong to the AGC family of basophilic protein Universite de Montreal, Montreal, Quebec, Canada. kinases (16). RSK has been shown to be hyperactivated in BRAF-mutated melanoma (17), and accordingly, inhibition of Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). RSK activity abrogates melanoma growth in mice (18). Although RSK regulates a number of substrates involved in cell growth and Current address for S.-P. Gravel: Faculty of Pharmacy, Universite de Montreal, Montreal, Quebec, Canada; current address for M. Savall: Institut Cochin, Uni- proliferation, a potential role for RSK signaling in cell metabolism versite Paris Descartes (Paris V); Centre National de la Recherche Scientifique remains unknown. (CNRS), UMR8104, Paris 75014, France; Institut National de la santeetdela One of the hallmarks of cancer is metabolic reprogramming, recherche medicale (INSERM), U1016, Paris 75014, France; and current address which facilitates the uptake and incorporation of nutrients needed for J. St-Pierre: Ottawa Institute of Systems Biology (OISB), Department of to support the continuous growth and proliferation of cancer cells Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, (19, 20). In particular, cancer cells have accelerated aerobic Ontario, Canada. glycolysis, which converts glucose to lactate regardless of oxygen Corresponding Author: Philippe P. Roux, UniversitedeMontr eal, Montreal, availability (21). Although much is known about the altered Quebec H3C 3J7, Canada. Phone: 514-343-6399; Fax: 514-343-5839; E-mail: glucose metabolism of cancer cells, more recent work has shown [email protected] that many of these changes are directly driven by oncogenes and doi: 10.1158/0008-5472.CAN-17-2215 tumor-suppressor genes (22). In melanoma, oncogenic BRAF is a Ó2018 American Association for Cancer Research. key player in metabolic reprogramming as it affects glucose www.aacrjournals.org 2191 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2215 Houles et al. metabolism in several ways (23). Activated BRAF downregu- after thawing. A375 stable cell lines were generated using lates expression of peroxisome proliferator–activated receptor g pBabe-puro–derived retroviral particles, and expressing cells were coactivator 1a (PGC-1a; refs. 24, 25), a transcriptional coactiv- selected using puromycin (2 mg/mL). HEK293 cells were trans- ator that promotes mitochondrial biogenesis and enhances oxi- fected by calcium phosphate precipitation as previously described dative phosphorylation (26), and stimulates glycolysis in part (37). Cells were grown for 24 hours after transfection and serum- by upregulating hypoxia-inducible factor 1 (HIF1) target genes starved overnight using serum-free DMEM where indicated. involved in glucose uptake and subsequent utilization along Starved cells were pretreated with PD184352 (10 mmol/L), the pathway (27, 28). One particular example is the upregulation LJH685 (10 mmol/L), BI-D1870 (10 mmol/L), PI-103 (1 mmol/L), of 6-phosphofructo-1-kinase (PFK-1; ref. 29), which is a key rapamycin (25 nmol/L), or Ku-0063794 (10 mmol/L; Biomol), regulatory enzyme that controls glycolytic flux (30). One of the where indicated, and stimulated with phorbol 12-myristate most potent activators of PFK-1 is fructose-2,6-bisphosphate 13-acetate (PMA; 100 ng/mL) or EGF (25 ng/mL) before being (Fru-2,6-P2), whose levels are controlled by the bifunctional harvested. Unless indicated otherwise, all drugs and growth 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) factors were purchased from Invitrogen. family of enzymes (PFKFB1–4; ref. 31). Although the expression of some PFK-2 family members has been shown to depend on RNA interference and viral infections HIF1 activity (32), a potential role for BRAF in the direct regu- Short hairpin RNA (shRNA)–mediated knockdown was lation of PFK-2 activity remains unknown. achieved using lentiviruses produced with vectors from the Mis- Recent phosphoproteomic studies performed in BRAF- sion TRC shRNA library (PFKFB2, TRCN37959, TRCN37960, mutated melanoma cells revealed PFKFB2 as a potential phos- TRCN37962; RSK1, TRCN470; RSK2, TRCN537). Cells were phorylation substrate of RSK (33, 34). Using various agonists infected in the presence of 4 mg/mL polybrene, and 3 days after and inhibitors of RAS/MAPK signaling, we found that PFKFB2 viral infection, A375 and WM164 cells were treated and selected is directly phosphorylated by RSK on sites that regulate its with 2 mg/mL puromycin. Retroviruses were produced in the function. RSK was found to promote glycolytic flux in mela- Phoenix cell line using the pLPC-puro vector to overexpress noma cells, which supports melanoma growth. ectopic PFKFB2 WT, S466/483A, or S466/483D mutants. Two days after infection, cells were selected with 2 mg/mL puromycin. Materials and Methods Immunoprecipitations and immunoblotting DNA constructs and recombinant proteins All cell lysates were prepared as previously described (38). The original plasmid encoding human PFKFB2 (pCR4-TOPO- Briefly, cells were washed 3 times with ice-cold PBS and lysed in PFKFB2) was obtained from the PlasmID Repository at Harvard BLB [10 mmol/L K3PO4, 1 mmol/L EDTA, 5 mmol/L EGTA, Medical School (Clone MGC:138310). This DNA construct was 10 mmol/L MgCl2, 50 mmol/L b-glycerophosphate, 0.5% used as template for generating 6Myc-tagged PFKFB2 in the Nonidet P-40, 0.1% Brij 35, 0.1% deoxycholic acid, 1 mmol/L fi pcDNA3 and pLPC backbones, as well as for the ampli cation sodium orthovanadate (Na3VO4), 1 mmol/L phenylmethylsul- of a C-terminal fragment of PFKFB2 (aa 431–505) that was fonyl fluoride, and a cOmplete protease inhibitor cocktail tablet inserted in pGEX-2T for bacterial