Anne-Christine Wong Te Fong

Histone deacetylase inhibition increases levels of choline kinase α and phosphocholine facilitating noninvasive imaging in human cancers.

Histone deacetylase (HDAC) inhibitors are currently approved for cutaneous T-cell lymphoma and are in mid-late stage trials for other cancers. The HDAC inhibitors LAQ824 and SAHA increase phosphocholine (PC) levels in human colon cancer cells and tumor xenografts as observed by magnetic resonance spectroscopy (MRS). In this study, we show that belinostat, an HDAC inhibitor with an alternative chemical scaffold, also caused a rise in cellular PC content that was detectable by (1)H and (31)P MRS in prostate and colon carcinoma cells. In addition, (1)H MRS showed an increase in branched chain amino acid and alanine concentrations. (13)C-choline labeling indicated that the rise in PC resulted from increased de novo synthesis and correlated with an induction of choline kinase α expression. Furthermore, metabolic labeling experiments with (13)C-glucose showed that differential glucose routing favored alanine formation at the expense of lactate production. Additional analysis revealed increases in the choline/water and phosphomonoester (including PC)/total phosphate ratios in vivo. Together, our findings provide mechanistic insights into the impact of HDAC inhibition on cancer cell metabolism and highlight PC as a candidate noninvasive imaging biomarker for monitoring the action of HDAC inhibitors.

Reduced Warburg Effect in Cancer Cells Undergoing Autophagy: Steady- State 1H-MRS and Real-Time Hyperpolarized 13C-MRS Studies

Autophagy is a highly regulated, energy dependent cellular process where proteins, organelles and cytoplasm are sequestered in autophagosomes and digested to sustain cellular homeostasis. We hypothesized that during autophagy induced in cancer cells by i) starvation through serum and amino acid deprivation or ii) treatment with PI-103, a class I PI3K/mTOR inhibitor, glycolytic metabolism would be affected, reducing flux to lactate, and that this effect may be reversible. We probed metabolism during autophagy in colorectal HT29 and HCT116 Bax knock-out cells using hyperpolarized 13C-magnetic resonance spectroscopy (MRS) and steady-state 1H-MRS. 24 hr PI103-treatment or starvation caused significant reduction in the apparent forward rate constant (kPL) for pyruvate to lactate exchange compared with controls in HT29 (100 μM PI-103: 82%, p = 0.05) and HCT116 Bax-ko cells (10 μM PI-103: 53%, p = 0.05; 20 μM PI-103: 42%, p<0.0001; starvation: 52%, p<0.001), associated with reduced lactate excretion and intracellular lactate in all cases, and unchanged lactate dehydrogenase (LDH) activity and increased NAD+/NADH ratio following PI103 treatment or decreased LDH activity and unchanged NAD+/NADH ratio following starvation. After 48 hr recovery from PI103 treatment, kPL remained below control levels in HT29 cells (74%, p = 0.02), and increased above treated values, but remained below 24 hr vehicle-treated control levels in HCT116 Bax-ko cells (65%, p = 0.004) both were accompanied by sustained reduction in lactate excretion, recovery of NAD+/NADH ratio and intracellular lactate. Following recovery from starvation, kPL was significantly higher than 24 hr vehicle-treated controls (140%, p = 0.05), associated with increased LDH activity and total cellular NAD(H). Changes in kPL and cellular and excreted lactate provided measureable indicators of the major metabolic processes accompanying starvation- and drug-induced autophagy. The changes are reversible, returning towards and exceeding control values on cellular recovery, which potentially identifies resistance. kPL (hyperpolarized 13C-MRS) and lactate (1H-MRS) provide useful biomarkers for the autophagic process, enabling non-invasive monitoring of the Warburg effect.

Evaluation of the combination of the dual m-TORC1/2 inhibitor vistusertib (AZD2014) and paclitaxel in ovarian cancer models

Activation of the PI3K/mTOR pathway has been shown to be correlated with resistance to chemotherapy in ovarian cancer. We aimed to investigate the effects of combining inhibition of mTORC1 and 2 using the mTOR kinase inhibitor vistusertib (AZD2014) with paclitaxel in in vitro and in vivo ovarian cancer models. The combination of vistusertib and paclitaxel on cell growth was additive in a majority of cell lines in the panel (n = 12) studied. A cisplatin- resistant model (A2780Cis) was studied in vitro and in vivo. We demonstrated inhibition of mTORC1 and mTORC2 by vistusertib and the combination by showing reduction in p-S6 and p-AKT levels, respectively. In the A2780CisR xenograft model compared to control, there was a significant reduction in tumor volumes (p = 0.03) caused by the combination and not paclitaxel or vistusertib alone. In vivo, we observed a significant increase in apoptosis (cleaved PARP measured by immunohistochemistry; p = 0.0003). Decreases in phospholipid and bioenergetic metabolites were studied using magnetic resonance spectroscopy and significant changes in phosphocholine (p = 0.01), and ATP (p = 0.04) were seen in tumors treated with the combination when compared to vehicle-control. Based on this data, a clinical trial evaluating the combination of paclitaxel and vistusertib has been initiated (NCT02193633). Interestingly, treatment of ovarian cancer patients with paclitaxel caused an increase in p-AKT levels in platelet-rich plasma and it was possible to abrogate this increase with the co-treatment with vistusertib in 4/5 patients: we believe this combination will benefit patients with ovarian cancer.

Abstract CT138: Translating preclinical observations to the clinic: Combination of the dual m-TORC1/2 inhibitor AZD2014 and paclitaxel in ovarian and lung cancer

Background: We have previously immunomagnetically separated cancer cells from ascites samples of patients with ovarian cancer who went on to receive chemotherapy. We reported that a raised p-S6K in cancer cells was associated with chemoresistance (Carden C, et al, Mol Cancer Ther 2012;11:1609-17). We hypothesized that combining chemotherapy with a novel dual m-TORC1/2 inhibitor would be effective in this setting. Methods: We studied apoptosis induced by paclitaxel (P), AZD2014 (A) and the combination (C) on 2 ovarian (A2780Cis, SKOV3), 2 lung (PC9, H520), 2 breast (MCF7, SKBR3) and 1 endothelial cell line (HUVEC) by quantifying cleaved-PARP using ELISA. We further investigated the effects of each drug and the combination on tumor growth and signal transduction (pSer473-AKT, p-S6) with a combination of P (20 mg/kg/week) and A (50 mg/kg/3 days/week) in 2 xenograft models (A2780Cis and H520). We also designed an investigator-initiated clinical trial of the combination of P 80 mg/m2/week in combination with an escalating dose of A administered twice a day in 2 schedules (2/7 and 3/7 started concomitantly with the P infusions). Radiological response was measured by RECIST and clinical benefit (CB) defined as progression at 14 weeks or later. Results: There was a major increase in apoptosis induction, as determined by c-PARP levels quantitation, in 3/6 cancer cell lines and the endothelial cell line HUVEC when A was added to P when compared to P alone utilizing concentrations of both drugs at GI50 and 5 x GI50 for 24 hrs. Additive growth inhibition was observed in both xenograft models after 2 weeks of combination treatment with tumor volume increased by 281 ± 98% with the combination (p Conclusions: The combination of P+A is effective in preclinical ovarian and lung cancer models, with additive growth inhibition and apoptosis seen. Promising drug combination antitumor activity has also been reported in heavily pre-treated patients with ovarian cancer and squamous NSCLC in an ongoing clinical trial. Citation Format: Parames Thavasu, Anne-Christine LF Wong Te Fong, Begona Jimenez Rodriguez, Bristi Basu, Alison Turner, Emma Hall, Timothy A. Yap, Susana Banerjee, Martin Leach, Johann S. de Bono, Yuen-Li Chung, Udai Banerji. Translating preclinical observations to the clinic: Combination of the dual m-TORC1/2 inhibitor AZD2014 and paclitaxel in ovarian and lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT138. doi:10.1158/1538-7445.AM2015-CT138

Metabolic biomarkers of response to the AKT inhibitor MK-2206 in pre-clinical models of human colorectal and prostate carcinoma

BackgroundAKT is commonly overexpressed in tumours and plays an important role in the metabolic reprogramming of cancer. We have used magnetic resonance spectroscopy (MRS) to assess whether inhibition of AKT signalling would result in metabolic changes that could potentially be used as biomarkers to monitor response to AKT inhibition.MethodsCellular and metabolic effects of the allosteric AKT inhibitor MK-2206 were investigated in HT29 colon and PC3 prostate cancer cells and xenografts using flow cytometry, immunoblotting, immunohistology and MRS.ResultsIn vitro treatment with MK-2206 inhibited AKT signalling and resulted in time-dependent alterations in glucose, glutamine and phospholipid metabolism. In vivo, MK-2206 resulted in inhibition of AKT signalling and tumour growth compared with vehicle-treated controls. In vivo MRS analysis of HT29 subcutaneous xenografts showed similar metabolic changes to those seen in vitro including decreases in the tCho/water ratio, tumour bioenergetic metabolites and changes in glutamine and glutathione metabolism. Similar phosphocholine changes compared to in vitro were confirmed in the clinically relevant orthotopic PC3 model.ConclusionThis MRS study suggests that choline metabolites detected in response to AKT inhibition are time and microenvironment-dependent, and may have potential as non-invasive biomarkers for monitoring response to AKT inhibitors in selected cancer types.

Abstract B56: Treatment-induced autophagy increases amino acid uptake and switches glucose addiction to amino acid catabolism in cancer

Abstracts: AACR Special Conference: Metabolism and Cancer; June 7-10, 2015; Bellevue, WA Background: Autophagy is a cellular catabolic degradation response to starvation or stress and the rerouting of cellular metabolism by autophagic cancer cells, to sustain cellular homeostasis during starvation or treatment-induced stress, remains poorly understood. We investigated the hypotheses that treatment-induced autophagy could prolong cancer cell survival, supported by adapted metabolism reversibly maintaining cellular homeostasis. Methods: We induced autophagy in human prostate (PC3) and colorectal (HT29, HCT116 WT (wild-type) and HCT116 Bax-ko (Bax-knock out)) cancer cells by 6 or 24 hours of amino-acid and serum deprivation (in Hanks balanced salt solution), or by 24 hours of treatment with PI103 (a phosphatidyl inositol-3 kinase inhibitor) or dichloroacetate (DCA, a pyruvate dehydrogenase kinase inhibitor). Cellular metabolism during starvation- or treatment-induced autophagy and subsequent recovery were examined by 1H- and 13C-magnetic resonance spectroscopy metabolomic studies. 13C-labeled glucose was used to assess metabolic flux. In vitro findings were verified in two corresponding colorectal xenograft models treated with PI103 or DCA. Results and Discussion: Increased expression of LC3II by western blots and the increased level of autophagosomes visualized by electron microscopy confirmed the induction of autophagy with minimal apoptosis and necrosis in HCT116 Bax-ko cells following starvation, DCA or PI103 treatment and in HCT116 WT, PC3 and HT29 cells following DCA treatment. PI103-induced autophagy prolonged cell survival, whereas starvation-induced autophagic cells eventually died. In PI103- or DCA-induced autophagy, metabolism was re-routed by i) reduced aerobic glycolysis with unchanged glucose uptake, increased cellular glucose levels (P<0.003) and reduced lactate excretion (P<0.0001); ii) increased uptake of branched-chain amino acids and glutamine (P<0.005), with a net accumulation of many intracellular amino acids and succinate (P<0.003), a TCA cycle intermediate. These metabolic alterations can prolong cancer cell survival during stress in a well-nourished environment by providing energy from amino-acid catabolism. Metabolic changes were reversed on recovery from treatment-induced autophagy. Increased levels of glutamine (P<0.01) and TCA-cycle intermediates (P<0.04) were also observed in DCA- and PI103-treated HT29 and HCT116-Bax-ko tumor xenografts, providing potential non-invasive biomarkers for treatment-induced autophagy. This work is supported by the CR-UK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) grants C1060/A10334 and C1060/A16464, NHS funding to the NIHR Biomedical Research Centre. Chang Gung Medical Foundation (Taiwan) grant CMRPG370441 and MRC-funded studentship (MRC119X). MOL is an NIHR Senior Investigator. We thank Alice Warley at the Kings College London Centre for Ultrastructural Imaging (CUI) for providing facilities for electron microscopy. Citation Format: Gigin Lin, Helen Troy, Gabriela Andrejeva, Anne-Christine LF Wong Te Fong, Dow-Mu Koh, Simon P. Robinson, Ian R. Judson, John R. Griffiths, Martin O. Leach, Yuen-Li Chung. Treatment-induced autophagy increases amino acid uptake and switches glucose addiction to amino acid catabolism in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B56.

Abstract 2897: Phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy

Autophagy is a cellular stress survival mechanism in which newly formed double-membrane vesicles target parts of the cytosol and organelles for lysosomal degradation to maintain cellular energy production. The source of the autophagosome membrane in starvation has been subject to much debate, but has not been investigated in the more clinically relevant setting of drug-induced autophagy where nutrients are available. By 1H-magnetic resonance spectroscopy (MRS) we show that autophagy, induced in human colorectal carcinoma cells HCT116 wt, HCT116 Bax-ko and HT29 by dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, by PI-103, a dual PI3K/mTOR inhibitor, or by starvation in medium lacking amino acids and growth factors, is associated with significant increases in membrane phospholipid phosphatidylcholine (PtdCho) (p≤0.02). 13-C labeled choline 13C-MRS revealed that these increases are due to de novo synthesis of PtdCho (p≤0.02) and are linked with increased expression of the rate-limiting enzyme, CDP:phosphocholine cytidylyltransferase (CCTα) in its active, membrane-bound form. Autophagic cells labeled with propargylcholine, a choline analog, had significant increase in propargylcholine phospholipid synthesis (p≤0.02) and vesicular structures consistent with autophagosomes when imaged by fluorescence confocal microscopy. Electron microscopy with immunogold labeling reveals the incorporation of newly synthesized propargylcholine phospholipids into the membranes of autophagic vacuoles and the digested material in autophagolysosomes, as well as endoplasmic reticulum, mitochondrial membranes and nuclear envelope as in control cells. To assess the role of PtdCho synthesis and CCTα in autophagy, we used Chinese hamster ovarian cells CHO-K1 MT58, with a temperature-sensitive mutation in the CCTα gene, resulting in reduced PtdCho synthesis at 40°C. PI-103 induced autophagy in wild-type CHO-K1 cells at both 33°C and 40°C. MT58 cells had normal autophagy induction with PI-103 at 33°C, but the autophagy marker LC3B-II was lower at 40°C than in the CHO-K1 cells. Loss of CCTα activity prevented PI-103-induced increase in PtdCho levels (p≤0.0003) and resulted in a reduced volume of autophagosomes in MT58 cells at 40°C (p≤0.01). Our evidence suggests that increased PtdCho synthesis in drug-induced autophagy provides membrane phospholipids for the growing autophagosomes and replaces phospholipids consumed from other organelles during autophagosome formation and degradation. Expression of CCTα, the rate-limiting enzyme of PtdCho synthesis, is increased in autophagy and its loss results in the inability of cells to maintain autophagosome formation. PtdCho synthesis might provide a new target for autophagy inhibition. Also, fluorescence imaging of propargylcholine labeling may offer a novel way to visualize newly formed autophagosome membranes in vitro. Citation Format: Gabriela Andrejeva, Gigin Lin, Harry G. Parkes, James Mui, Anne-Christine LF Wong Te Fong, Gema Vizcay-Barrena, Roland Fleck, Martin O. Leach, Yuen-Li Chung. Phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2897. doi:10.1158/1538-7445.AM2015-2897

Abstract 2451: Insulin-like growth factor-1 receptor (IGF-1R) inhibitors downregulate p53 expression and upregulate the Warburg effect in paediatric glioblastoma cells

Introduction: Previously we showed that Picropodophyllin (PPP) - an inhibitor of Insulin-Like Growth Factor-1 Receptor (IGF-1R) promotes the Warburg effect and this effect is associated with the downregulation of p53 after 24h of PPP-treatment. Activation of IKK-NF-κB, by loss of p53, has been reported to increase glycolysis in cancer (1). Aims: To understand the molecular mechanism that links p53 downregulation with increased glycolysis after PPP treatment, we studied the changes in glucose metabolism in 3 different IGF-1R inhibitors - PPP, OSI906 and NVP-AEW541 (NVP) and related these changes to IKK-NF-κB expression and other molecular markers. Methods: KNS42 cells were treated for 24 hours with 5xIC50 of either PPP (2.4µM), OSI906 (220µM) or NVP (40µM). Culture media from each treated group and controls were analyzed by 1H-MRS. Lactate dehydrogenase-A (LDH-A), p53, pAKT, pS6, MCT-1, MCT-4, Glut-1, pIKK and β-actin (loading control) were examined by western blot (WB). LDH enzyme activity was also measured in PPP- and NVP-treated cells. Results: After 24 hours of OSI906, NVP and PPP treatment, decreases of 30% (p Discussions and Conclusion: Our data indicated that the observed increased Warburg effect after IGF-1R inhibition is associated with increased Glut-1 expression, LDH activity and downregulation of p53 but the molecular mechanism is different in PPP when compared to OSI906 and NVP-treated cells. The increased Warburg effect after OSI906 and NVP treatment may be driven by the activation of the IKK-NF-κB pathway following the downregulation of p53, whereas upregulation of pS6 was seen in the PPP-treated cells. The activation of the mTOR-S6K pathway has been linked with the reduction of p53 expression (2). Further investigation is needed to examine why KNS42 cells have a different molecular response to PPP compared with OSI906 and NVP treatment. 1) Kawauchi et al., Nature Cell Biology 30(9): 1792-5, 2007. 2) Lai et al., EMBO 29: 2994-3006, 2010. Citation Format: Anne-Christine Wong Te Fong, Gabriela Andrejeva, Aleksandra Bielen, Chris Jones, John Griffiths, Martin O. Leach, Yuen-Li Chung. Insulin-like growth factor-1 receptor (IGF-1R) inhibitors downregulate p53 expression and upregulate the Warburg effect in paediatric glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2451. doi:10.1158/1538-7445.AM2014-2451

Abstract 5640: Picropodophyllin downregulates p53 and increases the Warburg effect in pediatric glioblastoma cells.

Picropodophyllin (PPP) is reported to be a potent and selective inhibitor of tyrosine phosphorylation of insulin-like growth factor-1 receptor (IGF-1R) [Girnita et al., Clin Cancer Res 12:1383-91, 2006]. We previously showed increases in glucose uptake and lactate production in paediatric glioblastoma KNS42 cells after 24h of PPP-treatment [Wong Te Fong et al., Proc Mol Tar Ther B61, 2012]. These findings are unexpected for IGF-1R inhibition. Upregulation of c-Myc or downregulation of p53 are known to upregulate glycolysis in cancer [DeBerardinis et al., Cell Metab 7:11-20, 2008a; Kawauchi et al., Nat Cell Bio 30(9): 1792-5, 2007]. In order to identify the molecular mechanism that drives our previously observed upregulation of glycolysis following PPP treatment, we examine the changes in glucose metabolism in PPP-treated cells over a 96h time-course and relate these metabolic changes to c-Myc and p53 expression. KNS42 cells were treated for 6, 24, 48 and 96h with 2.4μM (5xIC50) of PPP. Culture media from the PPP-treated and control cells were analyzed by 1H Magnetic Resonance Spectroscopy (MRS) to examine the effect of PPP on glucose uptake and lactate excretion. pErk, pAkt, c-Myc, p53 and β-actin (loading control) protein expressions were examined by Western blot (WB). No change in cell number was found after 6h of PPP treatment but ∼50% of PPP-treated cells remained by 24h when compared to controls (p We acknowledge the support received from the CRUK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) grant C1060/A10334, NHS funding to the NIHR Biomedical Research Centre. Citation Format: Anne-Christine LF Wong Te Fong, Chris Jones, John R. Griffiths, Martin O. Leach, Yuen-Li Chung. Picropodophyllin downregulates p53 and increases the Warburg effect in pediatric glioblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5640. doi:10.1158/1538-7445.AM2013-5640

Abstract B61: Picropodophyllin (PPP) increases glucose metabolism and lactate production in paediatric glioblastoma cells

Introduction: Picropodophyllin (PPP) is of interest as an anti-cancer agent, as it has been shown to induce growth inhibition and apoptosis in a variety of human cancer cells and xenografts. Recently, PPP was found to have dramatic antitumor effects in human glioblastoma cells and in subcutaneous and intracerebral xenografts and reported to be an insulin-like growth factor-1 receptor inhibitor; however, its full mechanisms of action remain unclear. Aims: To examine the effect of PPP on glucose metabolism in cancer cells and to use hyperpolarized [1-13C]pyruvate 13C-magnetic resonance spectroscopy (MRS) and conventional 1H-MRS to measure lactate formation/production in real-time and in steady state, respectively, in order to develop a non-invasive biomarker of response for PPP treatment in a pediatric glioblastoma cell line. Methods: Pediatric glioblastoma KNS42 cells were treated for 24 hours with 2.4μM (5×IC50) of PPP. Real-time 13C exchange from hyperpolarized [1-13C] pyruvate to lactate was performed by DNP-13C MRS. Culture media and extracts from the PPP-treated and control KNS42 cells were analyzed by 1H-MRS to examine the effect of PPP on glucose metabolism and lactate production. Lactate dehydrogenase (LDH) expression was examined by western blot and LDH activity was also measured enzymatically. Cellular protein content and size were also evaluated. Results and Discussion: The rate of real-time 13C exchange from pyruvate to lactate measured by DNP-13C MRS showed a significant increase in the PPP-treated group (P = 0.027 versus control group). These DNP changes in PPP-treated KNS42 cells are consistent with the significant increase in intracellular lactate (P = 0.003) and lactate excretion to the culture media (P < 0.001). This increase in lactate production is also consistent with significantly elevated LDH activity (P = 0.003) in the PPP-treated group. However, no change in LDH expression was observed in the PPP-treated group. Significant increases in intracellular glucose (P = 0.006) and glucose uptake (P = 0.008) were also found in PPP-treated KNS42 cells. These changes indicate an up-regulation of glucose metabolism following PPP treatment and a subsequent increase in lactate production. Twenty-four hours PPP treatment caused a significant decrease in cell number when compared to controls (∼40%, P = 0.001). No significant difference in cellular protein concentration and size were found in PPP-treated cells when compared to controls, indicating that the observed increases in glucose metabolism, lactate production and LDH activity are due to changes in cellular metabolism following PPP treatment and not from changes in cellular size or protein concentration. The increase in lactate production following PPP treatment found in this study is an unusual observation, as most cancer targeting-treatments cause a decrease in lactate production. Hence, the use of DNP and 13C MRS to measure lactate production could provide a more specific non-invasive biomarker of response for PPP treatment. Conclusions: PPP treatment resulted in increased glucose metabolism and lactate production in KNS42 cells, as shown by both real time and steady-state measurements. These changes have potential as specific biomarkers of response for PPP treatment. We acknowledge the support received from the CRUK and EPSRC Cancer Imaging Centre with the MRC and Department of Health (England) grant C1060/A10334, NHS funding to the NIHR Biomedical Research Centre.