Silencing of solute carrier family 13 member 5 disrupts energy homeostasis and inhibits proliferation of human hepatocarcinoma cells

Abstract

The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, plays a key role in importing citrate from the circulation into liver cells. Recent evidence has revealed that SLC13A5 deletion protects mice from high-fat diet–induced hepatic steatosis and that mutation of the SLC13A5 orthologues in Drosophila melanogaster and Caenorhabditis elegans promotes longevity. However, despite the emerging importance of SLC13A5 in energy homeostasis, whether perturbation of SLC13A5 affects the metabolism and malignancy of hepatocellular carcinoma is unknown. Here, we sought to determine whether SLC13A5 regulates hepatic energy homeostasis and proliferation of hepatoma cells. RNAi-mediated silencing of SLC13A5 expression in two human hepatoma cell lines, HepG2 and Huh7, profoundly suppressed cell proliferation and colony formation, and induced cell cycle arrest accompanied by increased expression of cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin B1. Furthermore, such suppressive effects were also observed on the growth of HepG2 cell-derived xenografts expressing SLC13A5-shRNA in nude mice. Metabolically, knockdown of SLC13A5 in HepG2 and Huh7 cells was associated with a decrease in intracellular levels of citrate, the ratio of ATP/ADP, phospholipid content, and ATP citrate lyase expression. Moreover, both in vitro and in vivo assays demonstrated that SLC13A5 depletion promotes activation of the AMP-activated protein kinase, which was accompanied by deactivation of oncogenic mechanistic target of rapamycin signaling. Together, our findings expand the role of SLC13A5 from facilitating hepatic energy homeostasis to influencing hepatoma cell proliferation and suggest a potential role of SLC13A5 in the progression of human hepatocellular carcinoma. The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, plays a key role in importing citrate from the circulation into liver cells. Recent evidence has revealed that SLC13A5 deletion protects mice from high-fat diet–induced hepatic steatosis and that mutation of the SLC13A5 orthologues in Drosophila melanogaster and Caenorhabditis elegans promotes longevity. However, despite the emerging importance of SLC13A5 in energy homeostasis, whether perturbation of SLC13A5 affects the metabolism and malignancy of hepatocellular carcinoma is unknown. Here, we sought to determine whether SLC13A5 regulates hepatic energy homeostasis and proliferation of hepatoma cells. RNAi-mediated silencing of SLC13A5 expression in two human hepatoma cell lines, HepG2 and Huh7, profoundly suppressed cell proliferation and colony formation, and induced cell cycle arrest accompanied by increased expression of cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin B1. Furthermore, such suppressive effects were also observed on the growth of HepG2 cell-derived xenografts expressing SLC13A5-shRNA in nude mice. Metabolically, knockdown of SLC13A5 in HepG2 and Huh7 cells was associated with a decrease in intracellular levels of citrate, the ratio of ATP/ADP, phospholipid content, and ATP citrate lyase expression. Moreover, both in vitro and in vivo assays demonstrated that SLC13A5 depletion promotes activation of the AMP-activated protein kinase, which was accompanied by deactivation of oncogenic mechanistic target of rapamycin signaling. Together, our findings expand the role of SLC13A5 from facilitating hepatic energy homeostasis to influencing hepatoma cell proliferation and suggest a potential role of SLC13A5 in the progression of human hepatocellular carcinoma. Metabolic reprogramming has long been regarded as a hallmark of cancer, through which cancer cells switch from oxidative phosphorylation to glycolysis for ATP production even in microenvironments with sufficient oxygen (1.Zheng J. Energy metabolism of cancer: glycolysis versus oxidative phosphorylation (Review).Oncol. Lett. 2012; 4: 1151-1157Crossref PubMed Scopus (541) Google Scholar, 2.Vander Heiden M.G. Cantley L.C. Thompson C.B. Understanding the Warburg effect: the metabolic requirements of cell proliferation.Science. 2009; 324: 1029-1033Crossref PubMed Scopus (10199) Google Scholar). To accommodate the challenge of rapid proliferation, cancer cell metabolism is remodeled to increase the biosynthesis of macromolecules including nucleotides, proteins, and lipids as building blocks of new cells (3.Obre E. Rossignol R. Emerging concepts in bioenergetics and cancer research: metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bioenergetic therapy.Int. J. Biochem. Cell Biol. 2015; 59: 167-181Crossref PubMed Scopus (99) Google Scholar, 4.Menendez J.A. Lupu R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis.Nat. Rev. Cancer. 2007; 7: 763-777Crossref PubMed Scopus (2030) Google Scholar). Although the precise mechanisms underlying this metabolic reprogramming remain elusive, malignant cells often have perturbed metabolism that allows for the accumulation of many metabolic intermediates facilitating the production of cellular building materials (4.Menendez J.A. Lupu R. 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Accumulating evidence suggests that citrate is involved in both physiological and pathophysiological processes including histone acetylation, insulin secretion, inflammation, cancer, neurological disorders, and non-alcoholic fatty liver disease (9.Icard P. Poulain L. Lincet H. Understanding the central role of citrate in the metabolism of cancer cells.Biochim. Biophys. Acta. 2012; 1825: 111-116PubMed Google Scholar, 10.Mycielska M.E. Patel A. Rizaner N. Mazurek M.P. Keun H. Patel A. Ganapathy V. Djamgoz M.B.A. Citrate transport and metabolism in mammalian cells: prostate epithelial cells and prostate cancer.Bioessays. 2009; 31: 10-20Crossref PubMed Scopus (103) Google Scholar). Cytosolic citrate is cleaved into acetyl-CoA and oxaloacetate by ATP citrate lyase (ACLY) (11.Chypre M. Zaidi N. Smans K. ATP-citrate lyase: a mini-review.Biochem. Biophys. Res. Commun. 2012; 422: 1-4Crossref PubMed Scopus (149) Google Scholar, 12.Lettieri Barbato D. Vegliante R. Desideri E. Ciriolo M.R. Managing lipid metabolism in proliferating cells: new perspective for metformin usage in cancer therapy.Biochim. Biophys. Acta. 2014; 1845: 317-324PubMed Google Scholar). Both acetyl-CoA and oxaloacetate are cytosolic precursors of multiple anabolic reactions important for de novo biosynthesis of fatty acids and steroids that are required for rapid proliferation, particularly in cancer cells (13.Zaidi N. Swinnen J.V. Smans K. ATP-citrate lyase: a key player in cancer metabolism.Cancer Res. 2012; 72: 3709-3714Crossref PubMed Scopus (320) Google Scholar). The intracellular level of citrate is tightly controlled by a balance between synthesis and transport. Mitochondrial citrate derived from the TCA cycle is transported to the cytosol via the citrate carrier (CIC), a member of the solute carrier transporter family (SLC25A1), and the majority of studies to date have focused on this citrate transporter (14.Palmieri F. The mitochondrial transporter family SLC25: identification, properties and physiopathology.Mol. Aspects Med. 2013; 34: 465-484Crossref PubMed Scopus (408) Google Scholar). For instance, limitation of citrate output from the mitochondria by CIC silencing is associated with decreased production of lipids and proinflammatory prostaglandins, as well as compromised adaptive cell survival responses (15.Infantino V. Convertini P. Cucci L. Panaro M.A. Di Noia M.A. Calvello R. Palmieri F. Iacobazzi V. The mitochondrial citrate carrier: a new player in inflammation.Biochem. J. 2011; 438: 433-436Crossref PubMed Scopus (254) Google Scholar, 16.O'Neill L.A. A critical role for citrate metabolism in LPS signalling.Biochem. J. 2011; 438: e5-e6Crossref PubMed Scopus (75) Google Scholar). In addition to mitochondrial synthesis and transport, cytosolic citrate can also be imported from the blood stream via a selective citrate uptake transporter SLC13A5, which is expressed predominantly in the liver (17.Inoue K. Zhuang L. Maddox D.M. Smith S.B. Ganapathy V. Structure, function, and expression pattern of a novel sodium-coupled citrate transporter (NaCT) cloned from mammalian brain.J. Biol. Chem. 2002; 277: 39469-39476Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 18.Inoue K. Fei Y.J. Zhuang L. Gopal E. Miyauchi S. Ganapathy V. Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates.Biochem. J. 2004; 378: 949-957Crossref PubMed Scopus (0) Google Scholar). As a member of the sodium dicarboxylate/sulfate cotransporter family, SLC13A5 recognizes and transports various dicarboxylate and tricarboxylate TCA intermediates with citrate as the K. Zhuang L. Ganapathy V. citrate genomic and transport Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar). SLC13A5 is expressed in the of and the uptake of citrate into from the the citrate is that of TCA intermediates that SLC13A5 a key physiological role in facilitating the use of citrate by the liver K. Fei Y.J. Zhuang L. Gopal E. Miyauchi S. Ganapathy V. Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates.Biochem. J. 2004; 378: 949-957Crossref PubMed Scopus (0) Google Scholar, E. Miyauchi S. S. Smith S.B. Ganapathy V. and features of NaCT, a sodium-coupled citrate transporter, in human and and cell J. 2007; PubMed Scopus Google Scholar, dicarboxylate and citrate from the 2014; PubMed Scopus (99) Google Scholar). The importance of SLC13A5 was observed in Drosophila melanogaster and Caenorhabditis in which expression of the SLC13A5 the of both S.P. by cotransporter in PubMed Scopus Google Scholar, Y.J. K. Zhuang L. K. Miyauchi S. Ganapathy V. of an citrate transporter, to and in Caenorhabditis J. 2004; PubMed Scopus Google Scholar). expression of SLC13A5 protects mice and from high-fat and insulin F. T.C. D. A. A. J. J. J. of the mammalian of and protects and insulin in Metab. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar, F. D. M. A. M.A. D.M. S. F. of hepatic steatosis and hepatic insulin by to the 2015; 7: PubMed Scopus Google Scholar). the of SLC13A5 expression was observed in non-alcoholic fatty liver disease and human and S. F. D.M. N. J. M. P. A. A. J. human and in hepatic lipid PubMed Scopus Google Scholar, L. H. H. J. S. M. H. SLC13A5 is a novel target of the and steatosis in human 2015; PubMed Scopus Google Scholar, F. A. S. S. J. as to lipid accumulation in 2015; PubMed Scopus Google Scholar). the emerging importance of SLC13A5 in hepatic energy homeostasis and metabolic disorders, whether the SLC13A5 affects the metabolism and malignant phenotype of cancer and of hepatocellular carcinoma cells in The was to the that SLC13A5 functions as a the proliferation of hepatoma cells by energy cell proliferation, colony formation, cell signaling as well as in vivo we demonstrated that of SLC13A5 the growth of hepatoma cells both in vitro and in of SLC13A5 expression in decreased levels of intracellular citrate, the ratio of ATP/ADP, and expression of SLC13A5 knockdown altered the of the AMP-activated protein kinase target of rapamycin To the role of SLC13A5 in the growth of hepatoma HepG2 and Huh7 cells were with and the as a As in A and both and the expression of SLC13A5 at the and protein silencing of SLC13A5 the proliferation of HepG2 and Huh7 cells in with the and in a and Moreover, colony assays revealed that depletion of SLC13A5 in HepG2 and Huh7 cells the and of on In a expression of SLC13A5 in and cells the growth of hepatoma cells the the growth of cells prostate cancer cell which SLC13A5, is by the of A and In knockdown of SLC13A5 in HepG2 and Huh7 cells has on the expression of the mitochondrial citrate transporter that is to SLC13A5 Together, suggest that SLC13A5 plays an important role in the growth and malignancy of human hepatoma cells such as HepG2 and Huh7 in which this is expressed we whether the cell cycle of hepatoma cells was by knockdown of revealed that silencing of SLC13A5 in HepG2 cells in with of cells in the versus of cells the cell in the and was decreased to versus and versus were observed and Huh7 cells that knockdown of SLC13A5 the growth of hepatoma through the of cell cycle arrest at the and revealed that knockdown of SLC13A5 in HepG2 and Huh7 cells is associated with decreased expression of cyclin and SLC13A5 silencing increased the expression of p21 in HepG2 such a is in Huh7 cells to the expression of p21 and that cyclin-dependent kinase to the observed cell cycle arrest in HepG2 a role in Huh7 cells. To determine whether SLC13A5 of hepatoma cell growth from cell and and propidium were to and in HepG2 and Huh7 cells with As a increased the of and silencing of SLC13A5 of A and of the and revealed that SLC13A5 knockdown is associated with of increased which be observed in the of as and that SLC13A5 knockdown HepG2 and Huh7 cell growth The of SLC13A5 is to citrate into liver cells and the intracellular energy balance K. Fei Y.J. Zhuang L. Gopal E. Miyauchi S. Ganapathy V. Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates.Biochem. J. 2004; 378: 949-957Crossref PubMed Scopus (0) Google Scholar). As demonstrated in A and selective depletion of SLC13A5 in of intracellular citrate and the ratio in HepG2 and Huh7 cells. this in cellular energy is associated with increased phosphorylation of and decreased of mTOR, two signaling that as cellular for energy homeostasis and oxidative the of Huh7 cells to levels of citrate in decreased phosphorylation of and an increased such were expression of SLC13A5 was and suggest that of intracellular citrate to SLC13A5 depletion to the of HepG2 and Huh7 cell proliferation through perturbation of the ratio and signaling. cytosolic citrate into which has been as the of de novo in F. D. Thompson C.B. ATP citrate lyase can cell Full Text Full Text PDF PubMed Scopus Google Scholar). The of expression in many cancer cells suggests that an for cancer (13.Zaidi N. Swinnen J.V. Smans K. ATP-citrate lyase: a key player in cancer metabolism.Cancer Res. 2012; 72: 3709-3714Crossref PubMed Scopus (320) Google Scholar). whether SLC13A5 knockdown affects the expression of in HepG2 and Huh7 cells. and that expression of was decreased at both the and protein levels SLC13A5 depletion A and In knockdown of SLC13A5 in HepG2 and Huh7 cells to intracellular in with cells with and that SLC13A5 knockdown expression and affects in To determine whether depletion of SLC13A5 also growth of hepatoma cells in HepG2 cells with SLC13A5-shRNA were into nude mice as growth was a of cell that the and in the were in the As expression of SLC13A5 was in xenografts of cyclin and expression was observed in derived from in with from cells and In with the in vitro knockdown of SLC13A5 also in increased phosphorylation of and decreased phosphorylation of in vivo that of SLC13A5 human hepatoma cell proliferation both in vivo and in vitro by energy metabolism and de novo lipid de novo is a key of proliferating cancer in which of cytosolic citrate into acetyl-CoA and oxaloacetate by the and for the biosynthesis of fatty acids and as building blocks for cell growth F. D. Thompson C.B. ATP citrate lyase can cell Full Text Full Text PDF PubMed Scopus Google Scholar, N. Swinnen J.V. Smans K. ATP citrate lyase knockdown growth arrest and through and 2012; PubMed Scopus Google Scholar). In the the level of cytosolic citrate is through from the TCA cycle in mitochondria and uptake from the circulation F. T.C. D. A. A. J. J. J. of the mammalian of and protects and insulin in Metab. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Palmieri F. A. S. E. The mitochondrial citrate transporter, CIC, is for mitochondrial 2012; PubMed Scopus Google Scholar). Recent studies that expression of SLC13A5 in mice and in D. melanogaster and elegans in a of metabolic that mice from and insulin in and S.P. by cotransporter in PubMed Scopus Google Scholar, Y.J. K. Zhuang L. K. Miyauchi S. Ganapathy V. of an citrate transporter, to and in Caenorhabditis J. 2004; PubMed Scopus Google Scholar, F. T.C. D. A. A. J. J. J. of the mammalian of and protects and insulin in Metab. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar, F. D. M. A. M.A. D.M. S. F. of hepatic steatosis and hepatic insulin by to the 2015; 7: PubMed Scopus Google Scholar, N. mitochondrial oxygen production and oxidative 2009; PubMed Scopus Google Scholar). the emerging of SLC13A5 in hepatic energy homeostasis, we sought to determine whether SLC13A5 plays a role in the growth of human hepatoma cells. In the we for the that depletion of SLC13A5 in HepG2 and Huh7 cells suppressed cell proliferation and colony formation, cell cycle Metabolically, knockdown of SLC13A5 to in intracellular levels of citrate, the phospholipid content, and expression in HepG2 and Huh7 cells. Moreover, both in vitro and in vivo assays demonstrated that depletion of SLC13A5 also the cellular energy sensor AMPK, accompanied by the deactivation of oncogenic signaling. Together, metabolic the growth of hepatocellular carcinoma cells of energy depletion has long been sought as an in the of (3.Obre E. Rossignol R. Emerging concepts in bioenergetics and cancer research: metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bioenergetic therapy.Int. J. Biochem. Cell Biol. 2015; 59: 167-181Crossref PubMed Scopus (99) Google Scholar, S. A. L. R. of cancer energy Res. 2009; PubMed Scopus Google Scholar). cloned from HepG2 SLC13A5 is expressed in HepG2 and Huh7 cell in with cells. that selective knockdown of SLC13A5 the growth of hepatoma cells the growth of cancer cells expression of SLC13A5, which suggests that this citrate uptake transporter is for the growth of a of hepatoma cells. SLC13A5 a for at a of liver of progression decreased cell proliferation cell that depletion of SLC13A5 suppressed the proliferation of HepG2 and Huh7 cells with arrest with decreased expression of cyclin is with a in which a of lipid HepG2 cell proliferation cell H. J. and activation of are associated with to of hepatic cell 2015; PubMed Scopus Google Scholar). we observed that knockdown of SLC13A5 in increased expression of p21 in HepG2 in Huh7 cells. is that the of the p21 is by protein S. J. Murphy M. of the by Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). that HepG2 and Huh7 cells of the the mutation in Huh7 cells to the level of p21 expression J. carcinoma and the role of 2012; PubMed Scopus Google Scholar). Although the effects of on the expression of p21 is of the of this is that of p21 by SLC13A5 silencing to the observed cell cycle arrest in HepG2 in Huh7 cells. AMPK, a central of cellular plays a key role in the of cancer cell growth in to energy of the ratio the activation of AMPK, which in and the of including acetyl-CoA and that are for lipid synthesis of hepatic A by phosphorylation and Biol. Chem. Full Text PDF PubMed Google Scholar). also cancer cell growth by which energy use and protein synthesis cancer progression AMP-activated protein kinase a novel genomic sensor in the Biol. 2014; PubMed Scopus Google Scholar). that knockdown of SLC13A5 is sufficient to decrease the intracellular levels of citrate and the ratio in HepG2 and Huh7 cells. the observed of and the of of the were in vivo with HepG2 cell-derived xenografts in nude that depletion of SLC13A5 hepatoma cell proliferation through on lipid metabolism and evidence that many in both and and a of are associated with the activation of K. S. liver by hepatic Res. 2012; PubMed Scopus Google Scholar, E. F. A. M. M. Di L. S. S. V. of activation in cancer with J. Cancer. 2014; PubMed Scopus Google Scholar). To the energy requirements for proliferation, cancer cells metabolic to increase and for is at the of this metabolic from glycolysis to lipid synthesis cytosolic citrate as and S. K. S. S. A. R. H. of ATP citrate lyase accumulation with altered fatty acid in cancer J. Cancer. 2014; PubMed Scopus Google Scholar). expression of has been in a of this an target with a of selective in in both cell and F. D. Thompson C.B. ATP citrate lyase can cell Full Text Full Text PDF PubMed Scopus Google Scholar, K. E. F. M. M. H. S. K. ATP citrate lyase: activation and in cell Res. PubMed Scopus Google Scholar). activation of is to be of the to expression by the of the Barbato D. Vegliante R. Desideri E. Ciriolo M.R. Managing lipid metabolism in proliferating cells: new perspective for metformin usage in cancer therapy.Biochim. Biophys. Acta. 2014; 1845: 317-324PubMed Google Scholar, S. E. M. M. and to hepatic steatosis and in Metab. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). the a that selective knockdown of can the phosphorylation and activation of in multiple cancer cell H. J. ATP-citrate lyase regulates cellular via an and J. 2015; PubMed Scopus Google Scholar). In the we that depletion of SLC13A5 increased the activation of and the expression of both in hepatoma cells in vitro and in a in of the between and ACLY, that energy induced by SLC13A5 knockdown a between activation and that to growth in human hepatoma cells. In the in this a novel role of SLC13A5 in the proliferation of a of human hepatocellular carcinoma cells. in vitro and in vivo we demonstrated that depletion of SLC13A5 to a in the growth of HepG2 and Huh7 cells with in de novo which from the activation of by the of and expression. our findings the role of SLC13A5 in hepatic lipid homeostasis and potential effects on the growth of human hepatoma and suggest that SLC13A5 a potential target for both metabolic and liver PI, and were from were by The used in this and and from Cell cell were from HepG2 cells were from the and Huh7 cells were from The of both was by HepG2 and Huh7 cells were in with and in at and SLC13A5 expression was by the into the of a which was by of of F. R. H. H. J. J. A novel is prostate cancer progression and promotes Res. 2009; PubMed Scopus Google Scholar). For SLC13A5 knockdown SLC13A5 which the were were into the and of the expression from was the L. J. M. L. 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J. and activation of are associated with to of hepatic cell 2015; PubMed Scopus Google Scholar). HepG2 and Huh7 cells with for were used as cells were a that were as cells. from HepG2 and Huh7 cells with SLC13A5-shRNA for from HepG2 and Huh7 cells with for was and the the of was on a in at for were with a to the HepG2 and Huh7 cells were cells in a with and in the by SLC13A5-shRNA the ratio was on the the The level of citrate in HepG2 and Huh7 cells with SLC13A5-shRNA was a citrate to the The phospholipid assays were with the the of the studies were by the and of the of of the were in at with a cycle and with and by SLC13A5-shRNA HepG2 cells were in on for cells were with of and in the and of nude mice to a K. P. S. M. S. P. 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