Patent ID: 12247973

EXAMPLES

To determine if cancer and normal cells take up extracellular citrate present at physiological concentrations, the inventors incubated different cell lines with [U-13C]citrate at 200 μM(24 h). Citrate uptake was assessed as the intracellular ratio of fully labelled13C to12C citrate in prostate (PC-3M), pancreatic (MiaPaCa-2) and gastric (TMK-1) cancer and in non-neoplastic breast (MCF10A) and prostate (PNT2-C2) cell lines. These studies show that cancer cells take up greater amounts of citrate than normal cells (FIG.1A). Depending on the conditions, up to a third of the total intracellular citrate pool in cancer cells is derived from uptake of extracellular citrate (FIG.1B); the strongest effects are observed in cells starved of glucose for 24 h and in cells grown for 72 h under hypoxia preceded by 24 h glucose deprivation, confirming active regulation of citrate uptake by cancer cells. We conclude that cancer cells take up extracellular citrate present at physiologically relevant levels, and this uptake is influenced by stress conditions.

We determined the amount of fully labelled intracellular glutamate and aspartate derived from either 25 mM [U-13C]-labelled glucose or 200 μM [U13C]-labelled citrate in prostate cancer PC-3M cells using HPLC-MS/MS (FIG.1C). As low glucose significantly affected citrate metabolism (FIG.1B), high glucose (25 mM) was used to sustain stable non-starvation conditions; since glutamine has been suggested to be the main source of citrate in cancer cells, it was also present in all experiments (2 mM). By determining the amount of glutamate and aspartate derived from labelled citrate as a percentage of these metabolites originating from labelled glucose, our results demonstrate that extracellular citrate is metabolised (FIG.1C). Interestingly, under hypoxia, the amount of fully labelled citrate derivatives increased. These results confirm that extracellular citrate is taken up in a controlled way and metabolised by cancer cells.

To exclude the possibility of intracellular Ca2+changes in the presence of extracellular citrate on the observed effects, intracellular Ca2+level was measured using live cell imaging in PC-3M cells loaded with Fura-2 (Supp.FIGS.1Aand B). No significant effect of extracellular citrate on intracellular Ca2+levels was detected, excluding citrate chelation of divalent cations as a possible non-specific action.

Since citrate cannot move freely through cellular membranes, its transport requires a carrier protein. Prostate cancer cells do not express any of the known plasma membrane di/tri-carboxylate transporters belonging to the SLC13 gene family13. Interestingly, PCR and Western blotting of PC-3M prostate cancer cells suggest a significant presence of the plasma membrane citrate carrier that was recently cloned from normal prostate PNT2-C2 cells14(pmCiC;FIG.1D). Sequencing of the PCR products confirms that PC-3M cells express pmCiC14. Western blot analysis of the plasma membrane proteins from prostate (PC-3M), colon (HT29), pancreatic (MiaPaCa2) and gastric (TMK1) cell lines indicate that expression of the pmCiC is not specific to only prostate cancer (FIG.1E).

To confirm that pmCiC is responsible for citrate uptake the inventors used siRNA to transiently silence pmCiC in PC-3M cells; indeed, a significantly reduced short-term (15 min) uptake of14C-labelled citrate is observed (FIG.1F). Intracellular content of the13C-citrate is also reduced in the presence of two different siRNAs in long term (24 h) experiments (Supp.FIG.2), confirming the function of pmCiC in extracellular citrate uptake by tumour cells. The pmCiC transporter determined to be expressed in cancer cells and responsible for citrate import has been shown previously to be present in normal prostate epithelial cells, with the function of exporting citrate into the lumen. Interestingly, this transporter has also been found to take up citrate when expressed in HEK cells, suggesting that the directional activity of the pmCiC depends on the cell type and plasma membrane composition14. We conclude that cancer cells express pmCiC in their plasma membrane and this protein is responsible for extracellular citrate uptake.

To establish the overall effects of extracellular citrate on cancer cell metabolism, changes in Krebs cycle and glycolysis were determined. We compared incorporation of13C from [U-13C]glucose into intermediates (HPLC-MS/MS) of the Krebs cycle in PC-3M cells in the presence or absence of extracellular citrate. Intracellular metabolite ratios were studied in prostate cancer cells grown under citrate-depleted conditions (dialysed serum) or with 200 μM citrate-supplemented media. Under normoxic conditions the incorporation of labelled carbons from glucose into fully labelled fumarate, malate, α-ketoglutarate and citrate is significantly decreased (13% to 41%) when cells are exposed to extracellular citrate (FIG.2A).

Changes in Krebs cycle activity were also determined by measuring the absolute amounts determined as a normalized response of the studied substrates (unlabelled and total13C depicting all substrates with any number of labelled carbons) in the presence or absence of unlabelled extracellular citrate (FIG.7). In the presence of unlabelled citrate, the total amount of intracellular12C-citrate, -α-ketoglutarate and -fumarate increased (14-26%). Interestingly, accumulation of fumarate is a reported characteristic of cancer cells15. Reciprocally, the inventors observed a 23% decrease in intracellular13C-labelled citrate with no change in the amount of labelled α-ketoglutarate and fumarate (FIG.7). These data confirm that extracellular citrate modifies Krebs cycle activity by increasing intracellular content of substrates derived from non-glucose sources. Using flow cytometry the inventors also determined that ROS levels in PC-3M cells grown with extracellular citrate were decreases by about 20%, compared to cells grown in citrate-depleted dialysed serum (FIG.2B); use of normal non-dialysed serum also reduces ROS levels. Extracellular citrate did not affect ROS synthesis in normal PNT2-C2 cells (FIG.2B). Therefore, decreased mitochondrial activity in the presence of extracellular citrate could affect processes such as apoptosis by reducing ROS synthesis.

The increase in unlabelled citrate and α-ketoglutarate, as well as fumarate accumulation, suggest that citrate uptake might partially relieve the requirement for mitochondria to supply citrate for cytoplasmic needs. Therefore, the inventors determined the amount of pmCiC in PC-3M cells in relation to the abundance of mitochondrial citrate transporter (mCiC), under different conditions (FIG.2C). Under normoxia, in the absence of extracellular citrate there is an increased abundance of mCiC in the mitochondria of PC-3M cells accompanied by a decrease in pmCiC, suggesting that cancer cells can function under different metabolic profiles also depending on the extracellular substrate availability. Interestingly, under hypoxia, the abundance of mCiC is unaffected by the absence of extracellular citrate, whilst there is a substantial increase of pmCiC (FIG.2C). This differential regulation could be explained by the fact that mitochondrial citrate synthesis cannot be increased in the absence of oxygen, thus inciting an increase in pmCiC. Importantly, expression of mCiC in normal PNT2-C2 cells was insensitive to the presence of extracellular citrate. Furthermore, expression of m- and pmCiC in PC-3M cell is similar under conditions with added citrate and FCS, consistent with the presence of ˜200 μM of citrate in serum (measured in media). These results suggest that extracellular citrate is able to influence mitochondrial activity in cancer cells.

Cancer cells take up extracellular glutamine to support their metabolism through reductive carboxylation, however, use of the Krebs cycle intermediates to synthesise glutamine in vivo by human glioblastoma has also been shown recently16. We examined the influence of extracellular citrate on glutamine metabolism in PC-3M cells by using [U-13C]glutamine. Cells treated with unlabelled extracellular citrate show a decreased ratio of13C incorporation into citrate and α-ketoglutarate from labelled glutamine (FIG.2D) accompanied by an increased ratio of the incorporation of13C from glutamine into aspartate and fully labelled proline. This suggests that extracellular citrate supports glutaminolysis, allowing for excess glutamate to be funnelled into proline biosynthesis. Decreased mitochondrial activity permits accumulation of aspartate derived from glutamine; aspartate is a substrate for generation of non-essential amino acids, which are crucial for cancer cell survival17.

To assess glycolysis the inventors measured (unlabelled) glucose uptake and lactate release in the media from PC-3M cells incubated±200 μM citrate for 24 h. Interestingly, while lactate production (measured as the absolute amount of lactate per media volume) is unaffected in PC-3M cells incubated with citrate, cells used ˜22% less glucose (FIG.3A). This effect is most likely related to increased conversion of pyruvate to lactate because of decreased mitochondrial citrate synthesis. This result supports our other data suggesting decreased Krebs cycle activity in the presence of extracellular citrate.

Measurements of free amino acids from the media in the presence of extracellular citrate showed increases in the release of glycine, alanine and glutamate-derived proline (11-12%;FIG.3B). These data support the overall hypothesis that extracellular citrate reduces citrate production needs, allowing for altered metabolism of available substrates. The concentration of other amino acids tested did not differ significantly (data not shown).

We further examined the effects of extracellular citrate on levels of intracellular free amino acids. PC-3M cells were grown in media supplemented with 25 mM13C6-labelled glucose±200 μM unlabelled citrate. In the presence of extracellular citrate a significant decrease is observed in13C incorporation from labelled glucose into glutamine, proline, ornithine and glutamate (a derivative of α-ketoglutarate) (FIG.3C). There are also significant increases in absolute amounts of both labelled and unlabelled arginine, as well as labelled asparagine (FIGS.8Aand B), while levels of unlabelled asparagine and labelled proline and ornithine are significantly decreased (FIGS.8Aand B). The levels of unlabelled proline, ornithine and glutamate remained unchanged.13C incorporation ratio into other measured amino acids is unaffected in the presence of extracellular citrate (data not shown). Decreased incorporation of13C derived from labelled glucose is consistent with the synthesis of amino acids from unlabelled sources (e.g. citrate). Amino acids such as arginine are necessary for cancer cell division and survival18.

An increase in the synthesis of amino acids in the presence of extracellular citrate prompted cell division testing. By microscopic cell counting (trypan blue exclusion) the inventors found that extracellular citrate increased PC-3M cell numbers by 20.3%±2.2% (P=0.003, n=5). These results were corroborated by flow cytometry-based cell division studies that revealed a sharp increase in the G2/M phase in PC-3M cells cultured with supplemented citrate, whilst there is a decrease in cells entering the non-dividing phase (G0/G1) (FIG.3D). Therefore, metabolic changes induced by extracellular citrate affect metabolism to an extent that impacts cellular processes such as cell division.

To confirm the relevance of the present findings to human carcinogenesis, expression of pmCiC in various human tissues was evaluated by immunohistochemistry (IHC). Benign normal prostatic epithelium showed pmCiC staining predominantly in the apical part of the cells (FIG.4A). pmCiC staining intensity in epithelial cells is increased in BPH (benign prostatic hyperplasia), correlating with elevated extracellular citrate levels associated with benign prostatic overgrowth19(FIG.4B). Importantly, diffuse and strong staining of pmCiC is also observed in cancer cells (FIG.4C) and correlated well with p63/Racemase/P504S cocktail staining20(double-staining method,FIG.4D). Benign prostatic epithelium with characteristic nuclear p63 positivity20(shown inFIG.4D) stained weakly with pmCiC (FIG.4CandFIG.9). In contrast, prostatic adenocarcinoma staining with pmCiC is stronger and more evenly dispersed (FIG.4CandFIG.9), correlating with cytoplasmic Racemase/P504S positivity (FIG.4D). Cancer cells also retain high expression levels of pmCiC at lymph node metastasis sites (FIGS.4Eand F). Immunohistochemical staining of pmCiC is also positive in other cancerous tissues, including pancreatic and gastric adenocarcinomas (FIG.10). Obtained in this study data suggest correlation between the intensity of pmCiC staining and tumour subtype (FIG.4andFIG.9,10) and the correlation of pmCiC expression with cancer agressiveness.

Our study shows that extracellular citrate at physiological concentrations affects overall cancer cell metabolism (as summarised inFIG.11). We have focused primarily on tumours originating from prostate because healthy prostatic cells are known to release a large amount of citrate, whilst citrate disappears from the gland when prostate growth becomes metastatic21. Changes in prostate cell metabolism (e.g. decreased expression of Zn2+transporters and increased activity of mitochondrial aconitase) have been suggested to account for extracellular citrate fluctuations22. However, our data offer an additional explanation for the decrease in extracellular citrate—citrate is taken up by malignant cells. This metabolic utilization of extracellular citrate: (1) reduces Krebs cycle activity, (2) allows for fumarate accumulation, (3) reduces glucose consumption, (4) increases crucial amino acid levels including arginine or proline, and (5) decreases ROS synthesis. Importantly, all of these observed changes have been previously shown to correlate with aggressiveness of cancer cells23,24, allowing for support of processes such as metabolism under stress (hypoxia) and cell proliferation; accordingly expression of the pmCiC correlates with the severity of cancer grade in the human cancers the inventors studied. We conclude that this plasma membrane transporter should be recognised in the search for potential novel targets in cancer therapy.

EXPERIMENTAL PROCEDURES

Cell Culture, PCR and Western Blotting

Cell lines were grown as described previouslyl13,14,25. The following chemicals were used: uniformly13C-labelled citric acid and glutamine and unlabelled citric acid (Sigma, St. Louis, MO, USA), uniformly13C-labelled glucose (Cambridge Isotope Laboratories, Andover, MA, USA), dialysed serum (PAN Biotech GmbH, Aidenbach, Germany) anti mCiC and pmCiC antibody14(mitochondrial citrate carrier, GenScript Inc., Piscataway, NJ, USA). Western blotting25and PCR14were performed as described before. Experimental media consisted of RPMI-1640, 5% dialysed serum, 2 mM glutamine, 25 mM glucose±200 μM, citrate unless otherwise stated. The incubation time varied between 24-72 h as specified. For the extraction of the plasma membrane protein the Plasma Membrane Protein Extraction Kit (Abcam, Cambridge, UK) was used. The purity of the extraction was verified by checking for the presence of mCiC and Tom40 in the extract.

Uptake Experiments and Metabolomics

Metabolites were extracted with 80% methanol and measured by HPLC-ESI-MS/MS on an AB SCI EX (Framingham, MA, USA) Q TRAP™ 4000 system. Multiple reaction monitoring (MRM) with one transition each for the unlabelled analyte and the labelled analogue(s) was used. Amino acids were derivatized using propyl chloroformate/propanol as recently described26. Krebs cycle intermediates were separated on a Phenomenex Luna NH2 (150×2 mm i.d., 3 μm, Torrence, CA, USA) column with a water (0.1% (v/v) formic acid)/acetonitrile gradient and ionized in negative mode. Lactate and glucose in the media were measured as previously described27.

Transient siRNA Transfections and Radiolabelled Citrate Uptake

14C citrate was purchased from Moravek Biochemicals (Brea, Canada) and experiments were performed as described14. For transient siRNA transfections, cells were preincubated with chloroquine for 2 h. This was followed by 24 h incubation with either siRNA or mock solution. Western blot analysis or uptake measurements were performed as described in other sections of Materials and Methods.

Immunohistochemistry

Human tissue was stained with the pmCiC antibody as described before14.

Flow Cytometry (ROS and Cell Cycle Measurement)

Studies were performed as before28. For cell cycle analysis incubation with RNase A was followed by propidium iodine staining (Sigma Aldrich, Germany). ROS production was detected with dihydrorhodamine 123 (Molecular Probes, Darmstadt, Germany). Analysis was performed using a FACSCanto (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer. At least 10,000 live cells were measured per sample. Dead cells were detected using the Aqua Live/Dead cell kit (Molecular Probes).

Proliferation

Cell numbers were assessed using a hemocytometer and trypan blue exclusion dye. Microscopic cell counts were performed by 3 independent investigators.

Calcium Imaging

The experiments were performed using a ZEISS live cell imaging setup (ZEISS, Jena, Germany). Fura-2/AM-loaded cells (2 μM, 45 min at 37° C.) were illuminated with light of 340 or 380 nm (BP 340/30 HE, BP 387/15 HE) using a fast wavelength switching and excitation device (Lambda DG-4, Sutter Instrument, Novato, CA, USA). Fluorescence was detected at 510 nm (BP 510/90 HE and FT 409) using an AxioCam MRm CCD camera (ZEISS). ZEN 2012 software (ZEISS) was used to control the hardware and acquire data.

Calculations and Statistics

Percentage differences denote change of the experimental values as compared to the control data (considered to be 100%). Data are presented as mean±SD, number of replicates n≥5. Statistical significance was assessed using a two-tailed t-test.

Gluconate-inhibitor of pmCiC

Several lines of evidence presented below indicate gluconate as an inhibitor of pmCiC.

1. pmCiC expression in oocytes—two electrodes voltage clamp (FIG.13)

We have expressed pmCiC in oocytes and induced citrate inward current by introducing citrate into extracellular media. When applied extracellularly in the presence of gluconate, citrate-induced current is significantly decreased. This reduction was irreversible, as subsequent application of citrate after gluconate removal from the media was not able to restore the primary response.

2. Patch clamp on human prostate cancer PC-3M and benign PNT2-C2 cells (FIG.14)

Extracellular application of citrate induced inward current in both cell lines as described earlier. Here we show that similar to the case of oocytes when applied in the presence of gluconate, citrate-induced current is decreased. Importantly, repeated application of gluconate results in further citrate-induced current reduction. As in the case of oocytes, gluconate inhibition of citrate-induced current was irreversible. Repeated application of citrate on PC-3M cells has been previously shown not to cause any reduction in citrate current (Mycielska et al., 2005).

3. Effects of extracellular gluconate on pmCiC mRNA expression (FIG.15)

We have studied the effect of gluconate in the extracellular media supplemented with citrate on the expression changes of pmCiC and mCiC as compared to the control conditions where the cells were incubated with extracellular citrate only. The Human metastatic prostate PC-3M cells incubated in media supplemented with 5 mM gluconate have shown significantly increased pmCIC and mCiC mRNA expression (FIG.15). This result would suggest a cellular adaptation to compensate for the loss of citrate intake due to gluconate inhibition. On the other hand, there was a significant mCiC mRNA increase, which would indicate increased mitochondrial activity to maintain intracellular citrate level.

4. Effect of gluconate on the survival of human prostate cancer PC-3M cells (16)

Results show that 200 μM of extracellular citrate increases survival of cancer cells incubated under stress conditions (in serum-free media with 0.5 g/L gluc). Importantly, in the presence of gluconate, this protective effect of extracellular citrate is abolished and the survival of cells is the same as in the absence of extracellular citrate. Importantly, gluconate has no effect on cell survival in the absence of extracellular citrate.

LITERATURE

1. Vander Heiden, M. G., Cantley, L. C. & Thompson, C. B. Understanding the Warburg effect: the metabolic requirements of cell proliferation.Science324, 1029-1033 (2009).2. Currie, E., Schulze, A., Zechner, R., Walther, T. C. & Farese, R. V. Jr. Cellular Fatty Acid metabolism and cancer.Cell. Metab.18, 153-161 (2013).3. Liu, Y. Fatty acid oxidation is a dominant bioenergetic pathway in prostate cancer.Prostate Cancer Prostatic Dis.9, 230-234 (2006).4. Holla, V. R, Wu, H., Shi, Q., Menter, D.G & DuBois, R. N. Nuclear orphan receptor NR4A2 modulates fatty acid oxidation pathways in colorectal cancer.J. Biol. Chem.286, 30003-30009 (2011).5. Linher-Melville, K. et al. Establishing a relationship between prolactin and altered fatty acid β-oxidation via carnitine palmitoyl transferase 1 in breast cancer cells.BMC Cancer4, 11-56 (2011).6. Migita, T. et al. ATP citrate lyase: activation and therapeutic implications in non-small cell lung cancer.Cancer Res.68, 8547-8554 (2008).7. Frezza, C. & Gottlieb, E. Mitochondria in cancer: not just innocent bystanders.Semin. Cancer Biol.19, 4-11 (2009).8. Metallo, C. M. et al. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia.Nature481, 380-384 (2011).9. Rocha, C. M. et al. Metabolic signatures of lung cancer in biofluids: NMR-based metabonomics of blood plasma.J. Proteome Res.10, 4314-4324 (2011).10. Cao, M., Zhao, L., Chen, H., Xue, W. & Lin, D. NMR-based metabolomic analysis of human bladder cancer.Anal. Sci.28, 451-456 (2012).11. Zhang, L. et al. Distinguishing pancreatic cancer from chronic pancreatitis and healthy individuals by (1)H nuclear magnetic resonance-based metabonomic profiles.Clin. Biochem.45, 1064-1069 (2012).12. Clyne, M. Prostate cancer: Biopsy citrate concentration could predict prostate cancer growth rate.Nat. Rev. Urol.9, 123. (2012).13. Mycielska, M. E., Palmer, C. P., Brackenbury, W. J. & Djamgoz, M. B. Expression of Na+-dependent citrate transport in a strongly metastatic human prostate cancer PC-3M cell line: regulation by voltage-gated Na+ channel activity.J. Physiol.563, 393-408 (2005).14. Mazurek, M. P. et al. Molecular origin of plasma membrane citrate transporter in human prostate epithelial cells.EMBO Rep.11, 431-437 (2010).15. Ratcliffe, P. J. Fumarate hydratase deficiency and cancer: activation of hypoxia signaling? Cancer Cell. 11, 303-305. (2007).16. Marin-Valencia, I. et al. Analysis of tumor metabolism reveals mitochondrial glucose oxidation in genetically diverse human glioblastomas in the mouse brain in vivo.Cell Metab.15, 827-837 (2012).17. Son, J. et al. Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway.Nature496, 101-105 (2013).18. Jang, S. W. et al. Serine/arginine protein-specific kinase 2 promotes leukemia cell proliferation by phosphorylating acinus and regulating cyclin A1.Cancer Res.68, 4559-4570 (2008).19. Costello, L. C. & Franklin, R. B. Citrate metabolism of normal and malignant prostate epithelial cells.Urology50, 3-12 (1997).20. Evans, A. J. Alpha-methylacyl CoA racemase (P504S): overview and potential uses in diagnostic pathology as applied to prostate needle biopsies.J. Clin. Pathol.56, 892-897 (2003).21. Serkova, N. J. et al. The metabolites_citrate, myo-inositol, and spermine are potential age-independent markers of prostate cancer in human expressed prostatic secretions. Prostate 68, 620-628 (2008).22. Singh, K. K., Desouki, M. M., Franklin, R. B. & Costello, L. C. Mitochondrial aconitase and citrate metabolism in malignant and nonmalignant human prostate tissues.Mol. Cancer4, 5-14 (2006).23. Cardaci, S. & Ciriolo, M. R. TCA Cycle Defects and Cancer: When Metabolism Tunes Redox State. Int. J. Cell Biol. 2012, 161837 (2012). 24. Cuperlovic-Culf, M., Culf, A. S., Touaibia, M. & Lefort, N. Targeting the latest hallmark of cancer: another attempt at ‘magic bullet’ drugs targeting cancers' metabolic phenotype.Future Oncol.8, 1315-1330 (2012).25. Lang, S. A. et al. Mammalian target of rapamycin is activated in human gastric cancer and serves as a target for therapy in an experimental model. Int. J. Cancer. 120, 1803-1810 (2007).26. Van der Goot, A. T. et al. Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation.PNAS109, 14912-14917 (2012).27. Dettmer, K. et al. Distinct metabolic differences between various human cancer and primary cells.Electrophoresis34, 2836-2847 (2013).28. Lantow, M., Viergutz, T., Weiss, D. G. & Simkó. M. Comparative study of cell cycle kinetics and induction of apoptosis or necrosis after exposure of human Mono Mac 6 cells to radiofrequency radiation.Radiat. Res.166, 539-543 (2006).