Abstract
In this study, we investigated the effects and the underlying molecular mechanisms of the multi-kinase inhibitor sorafenib in a panel of breast cancer cell lines. Sorafenib inhibited cell proliferation and induced apoptosis through the mitochondrial pathway. These effects were neither correlated with modulation of MAPK and AKT pathways nor dependent on the ERα status. Sorafenib promoted an early perturbation of mitochondrial function, inducing a deep depolarization of mitochondrial membrane, associated with drop of intracellular ATP levels and increase of ROS generation. As a response to this stress condition, the energy sensor AMPK was rapidly activated in all the cell lines analyzed. In MCF-7 and SKBR3 cells, AMPK enhanced glucose uptake by up-regulating the expression of GLUT-1 glucose transporter, as also demonstrated by AMPKα1 RNA interference, and stimulated aerobic glycolysis thus increasing lactate production. Moreover, the GLUT-1 inhibitor fasentin blocked sorafenib-induced glucose uptake and potentiated its cytotoxic activity in SKBR3 cells. Persistent activation of AMPK by sorafenib finally led to the impairment of glucose metabolism both in MCF-7 and SKBR3 cells as well as in the highly glycolytic MDA-MB-231 cells, resulting in cell death. This previously unrecognized long-term effect of sorafenib was mediated by AMPK-dependent inhibition of the mTORC1 pathway. Suppression of mTORC1 activity was sufficient for sorafenib to hinder glucose utilization in breast cancer cells, as demonstrated by the observation that the mTORC1 inhibitor rapamycin induced a comparable down-regulation of GLUT-1 expression and glucose uptake. The key role of AMPK-dependent inhibition of mTORC1 in sorafenib mechanisms of action was confirmed by AMPKα1 silencing, which restored mTORC1 activity conferring a significant protection from cell death. This study provides insights into the molecular mechanisms driving sorafenib anti-tumoral activity in breast cancer, and supports the need for going on with clinical trials aimed at proving the efficacy of sorafenib for breast cancer treatment.
Similar content being viewed by others
References
Kane RC, Farrell AT, Saber H, Tang S, Williams G, Jee JM, Liang C, Booth B, Chidambaram N, Morse D, Sridhara R, Garvey P, Justice R, Pazdur R (2006) Sorafenib for the treatment of advanced renal cell carcinoma. Clin Cancer Res 12(24):7271–7278
Kane RC, Farrell AT, Madabushi R, Booth B, Chattopadhyay S, Sridhara R, Justice R, Pazdur R (2009) Sorafenib for the treatment of unresectable hepatocellular carcinoma. Oncologist 14(1):95–100
Hatzivassiliou G, Song K, Yen I, Brandhuber BJ, Anderson DJ, Alvarado R, Ludlam MJ, Stokoe D, Gloor SL, Vigers G, Morales T, Aliagas I, Liu B, Sideris S, Hoeflich KP, Jaiswal BS, Seshagiri S, Koeppen H, Belvin M, Friedman LS, Malek S (2010) RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464(7287):431–435
Poulikakos PI, Zhang C, Bollag G, Shokat KM, Rosen N (2010) RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464(7287):427–430
Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, Chen C, Zhang X, Vincent P, McHugh M, Cao Y, Shujath J, Gawlak S, Eveleigh D, Rowley B, Liu L, Adnane L, Lynch M, Auclair D, Taylor I, Gedrich R, Voznesensky A, Riedl B, Post LE, Bollag G, Trail PA (2004) BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 64(19):7099–7109
Plaza-Menacho I, Mologni L, Sala E, Gambacorti-Passerini C, Magee AI, Links TP, Hofstra RM, Barford D, Isacke CM (2007) Sorafenib functions to potently suppress RET tyrosine kinase activity by direct enzymatic inhibition and promoting RET lysosomal degradation independent of proteasomal targeting. J Biol Chem 282(40):29230–29240
Bonelli MA, Fumarola C, Alfieri RR, La Monica S, Cavazzoni A, Galetti M, Gatti R, Belletti S, Harris AL, Fox SB, Evans DB, Dowsett M, Martin LA, Bottini A, Generali D, Petronini PG (2010) Synergistic activity of letrozole and sorafenib on breast cancer cells. Breast Cancer Res Treat 124(1):79–88
Huynh H, Ngo VC, Koong HN, Poon D, Choo SP, Thng CH, Chow P, Ong HS, Chung A, Soo KC (2009) Sorafenib and rapamycin induce growth suppression in mouse models of hepatocellular carcinoma. J Cell Mol Med 13(8B):2673–2683
Yu C, Bruzek LM, Meng XW, Gores GJ, Carter CA, Kaufmann SH, Adjei AA (2005) The role of Mcl-1 downregulation in the proapoptotic activity of the multikinase inhibitor BAY 43-9006. Oncogene 24(46):6861–6869
Ding Q, Huo L, Yang JY, Xia W, Wei Y, Liao Y, Chang CJ, Yang Y, Lai CC, Lee DF, Yen CJ, Chen YJ, Hsu JM, Kuo HP, Lin CY, Tsai FJ, Li LY, Tsai CH, Hung MC (2008) Down-regulation of myeloid cell leukemia-1 through inhibiting Erk/Pin 1 pathway by sorafenib facilitates chemosensitization in breast cancer. Cancer Res 68(15):6109–6117
Fiume L, Manerba M, Vettraino M, Di Stefano G (2011) Effect of sorafenib on the energy metabolism of hepatocellular carcinoma cells. Eur J Pharmacol 670(1):39–43
Bull VH, Rajalingam K, Thiede B (2012) Sorafenib-induced mitochondrial complex I inactivation and cell death in human neuroblastoma cells. J Proteome Res 11(3):1609–1620
Baselga J, Segalla JG, Roche H, Del Giglio A, Pinczowski H, Ciruelos EM, Filho SC, Gomez P, Van Eyll B, Bermejo B, Llombart A, Garicochea B, Duran MA, Hoff PM, Espie M, de Moraes AA, Ribeiro RA, Mathias C, Gil Gil M, Ojeda B, Morales J, Kwon Ro S, Li S, Costa F (2012) Sorafenib in combination with capecitabine: an oral regimen for patients with HER2-negative locally advanced or metastatic breast cancer. J Clin Oncol 30(13):1484–1491
Hudis C, Tauer KW, Hermann G, et al (2011) Sorafenib (SOR) plus chemotherapy (CRx) for patients (pts) with advanced (adv) breast cancer (BC) previously treated with bevacizumab (BEV). J Clin Oncol 29(suppl; abstr 1009)
Gradishar WJ, Kaklamani V, Sahoo TP, Lokanatha D, Raina V, Bondarde S, Jain M, Ro SK, Lokker NA, Schwartzberg L (2013) A double-blind, randomised, placebo-controlled, phase 2b study evaluating sorafenib in combination with paclitaxel as a first-line therapy in patients with HER2-negative advanced breast cancer. Eur J Cancer 49(2):312–322
Isaacs C, Herbolsheimer P, Liu MC, Wilkinson M, Ottaviano Y, Chung GG, Warren R, Eng-Wong J, Cohen P, Smith KL, Creswell K, Novielli A, Slack R (2011) Phase I/II study of sorafenib with anastrozole in patients with hormone receptor positive aromatase inhibitor resistant metastatic breast cancer. Breast Cancer Res Treat 125(1):137–143
La Monica S, Galetti M, Alfieri RR, Cavazzoni A, Ardizzoni A, Tiseo M, Capelletti M, Goldoni M, Tagliaferri S, Mutti A, Fumarola C, Bonelli M, Generali D, Petronini PG (2009) Everolimus restores gefitinib sensitivity in resistant non-small cell lung cancer cell lines. Biochem Pharmacol 78(5):460–468
Fumarola C, La Monica S, Alfieri RR, Borra E, Guidotti GG (2005) Cell size reduction induced by inhibition of the mTOR/S6 K-signaling pathway protects Jurkat cells from apoptosis. Cell Death Differ 12(10):1344–1357
Zhao Y, Wieman HL, Jacobs SR, Rathmell JC (2008) Mechanisms and methods in glucose metabolism and cell death. Methods Enzymol 442:439–457
Ashcroft SJ, Weerasinghe LC, Bassett JM, Randle PJ (1972) The pentose cycle and insulin release in mouse pancreatic islets. Biochem J 126(3):525–532
Zhao W, Zhang T, Qu B, Wu X, Zhu X, Meng F, Gu Y, Shu Y, Shen Y, Sun Y, Xu Q (2011) Sorafenib induces apoptosis in HL60 cells by inhibiting Src kinase-mediated STAT3 phosphorylation. Anticancer Drugs 22(1):79–88
Will Y, Dykens JA, Nadanaciva S, Hirakawa B, Jamieson J, Marroquin LD, Hynes J, Patyna S, Jessen BA (2008) Effect of the multitargeted tyrosine kinase inhibitors imatinib, dasatinib, sunitinib, and sorafenib on mitochondrial function in isolated rat heart mitochondria and H9c2 cells. Toxicol Sci 106(1):153–161
Coriat R, Nicco C, Chereau C, Mir O, Alexandre J, Ropert S, Weill B, Chaussade S, Goldwasser F, Batteux F (2012) Sorafenib-induced hepatocellular carcinoma cell death depends on reactive oxygen species production in vitro and in vivo. Mol Cancer Ther 11(10):2284–2293
Valabrega G, Capellero S, Cavalloni G, Zaccarello G, Petrelli A, Migliardi G, Milani A, Peraldo-Neia C, Gammaitoni L, Sapino A, Pecchioni C, Moggio A, Giordano S, Aglietta M, Montemurro F (2011) HER2-positive breast cancer cells resistant to trastuzumab and lapatinib lose reliance upon HER2 and are sensitive to the multitargeted kinase inhibitor sorafenib. Breast Cancer Res Treat 130(1):29–40
Heravi M, Tomic N, Liang L, Devic S, Holmes J, Deblois F, Radzioch D, Muanza T (2012) Sorafenib in combination with ionizing radiation has a greater anti-tumour activity in a breast cancer model. Anticancer Drugs 23(5):525–533
Tran MA, Smith CD, Kester M, Robertson GP (2008) Combining nanoliposomal ceramide with sorafenib synergistically inhibits melanoma and breast cancer cell survival to decrease tumor development. Clin Cancer Res 14(11):3571–3581
Wilhelm SM, Adnane L, Newell P, Villanueva A, Llovet JM, Lynch M (2008) Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling. Mol Cancer Ther 7(10):3129–3140
O’Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D, Lane H, Hofmann F, Hicklin DJ, Ludwig DL, Baselga J, Rosen N (2006) mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 66(3):1500–1508
Meric-Bernstam F, Akcakanat A, Chen H, Do KA, Sangai T, Adkins F, Gonzalez-Angulo AM, Rashid A, Crosby K, Dong M, Phan AT, Wolff RA, Gupta S, Mills GB, Yao J (2012) PIK3CA/PTEN mutations and Akt activation as markers of sensitivity to allosteric mTOR inhibitors. Clin Cancer Res 18(6):1777–1789
Rahmani M, Davis EM, Crabtree TR, Habibi JR, Nguyen TK, Dent P, Grant S (2007) The kinase inhibitor sorafenib induces cell death through a process involving induction of endoplasmic reticulum stress. Mol Cell Biol 27(15):5499–5513
Sanchez-Hernandez I, Baquero P, Calleros L, Chiloeches A (2012) Dual inhibition of (V600E)BRAF and the PI3K/AKT/mTOR pathway cooperates to induce apoptosis in melanoma cells through a MEK-independent mechanism. Cancer Lett 314(2):244–255
Ulivi P, Arienti C, Amadori D, Fabbri F, Carloni S, Tesei A, Vannini I, Silvestrini R, Zoli W (2009) Role of RAF/MEK/ERK pathway, p-STAT-3 and Mcl-1 in sorafenib activity in human pancreatic cancer cell lines. J Cell Physiol 220(1):214–221
Llobet D, Eritja N, Yeramian A, Pallares J, Sorolla A, Domingo M, Santacana M, Gonzalez-Tallada FJ, Matias-Guiu X, Dolcet X (2010) The multikinase inhibitor Sorafenib induces apoptosis and sensitises endometrial cancer cells to TRAIL by different mechanisms. Eur J Cancer 46(4):836–850
Cervello M, Bachvarov D, Lampiasi N, Cusimano A, Azzolina A, McCubrey JA, Montalto G (2012) Molecular mechanisms of sorafenib action in liver cancer cells. Cell Cycle 11(15):2843–2855
Cardaci S, Filomeni G, Ciriolo MR (2012) Redox implications of AMPK-mediated signal transduction beyond energetic clues. J Cell Sci 125(Pt 9):2115–2125
Marsin AS, Bertrand L, Rider MH, Deprez J, Beauloye C, Vincent MF, Van den Berghe G, Carling D, Hue L (2000) Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia. Curr Biol 10(20):1247–1255
Almeida A, Moncada S, Bolanos JP (2004) Nitric oxide switches on glycolysis through the AMP protein kinase and 6-phosphofructo-2-kinase pathway. Nat Cell Biol 6(1):45–51
Hao WS, Chang CPB, Tsao CC, Xu J (2010) Oligomycin-induced bioenergetic adaptation in cancer cells with heterogeneous bioenergetic organization. J Biol Chem 285(17):12647–12654
Wu SB, Wei YH (2012) AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: implication of the cell survival in mitochondrial diseases. Biochim Biophys Acta 1822(2):233–247
Riganti C, Gazzano E, Polimeni M, Costamagna C, Bosia A, Ghigo D (2004) Diphenyleneiodonium inhibits the cell redox metabolism and induces oxidative stress. J Biol Chem 279(46):47726–47731
Duvel K, Yecies JL, Menon S, Raman P, Lipovsky AI, Souza AL, Triantafellow E, Ma Q, Gorski R, Cleaver S, Vander Heiden MG, MacKeigan JP, Finan PM, Clish CB, Murphy LO, Manning BD (2010) Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol Cell 39(2):171–183
Shaw RJ (2006) Glucose metabolism and cancer. Curr Opin Cell Biol 18(6):598–608
Shen YC, Ou DL, Hsu C, Lin KL, Chang CY, Lin CY, Liu SH, Cheng AL (2013) Activating oxidative phosphorylation by a pyruvate dehydrogenase kinase inhibitor overcomes sorafenib resistance of hepatocellular carcinoma. Br J Cancer 108(1):72–81
Acknowledgments
We thank Bayer HealthCare Pharmaceuticals for providing sorafenib and A.VO.PRO.RI.T., Parma, Italy for its support.
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Fumarola, C., Caffarra, C., La Monica, S. et al. Effects of sorafenib on energy metabolism in breast cancer cells: role of AMPK–mTORC1 signaling. Breast Cancer Res Treat 141, 67–78 (2013). https://doi.org/10.1007/s10549-013-2668-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10549-013-2668-x