L. Elizabeth Jackson

Identification of magnetic resonance detectable metabolic changes associated with inhibition of phosphoinositide 3-kinase signaling in human breast cancer cells

Phosphoinositide 3-kinase (PI3K) is an attractive target for novel mechanism-based anticancer treatment. We used magnetic resonance (MR) spectroscopy (MRS) to detect biomarkers of PI3K signaling inhibition in human breast cancer cells. MDA-MB-231, MCF-7, and Hs578T cells were treated with the prototype PI3K inhibitor LY294002, and the 31P MR spectra of cell extracts were monitored. In every case, LY294002 treatment was associated with a significant decrease in phosphocholine levels by up to 2-fold (P < 0.05). In addition, a significant increase in glycerophosphocholine levels by up to 5-fold was also observed (P ≤ 0.05), whereas the content of glycerophosphoethanolamine, when detectable, did not change significantly. Nucleotide triphosphate levels did not change significantly in MCF-7 and MDA-MB-231 cells but decreased by ∼1.3-fold in Hs578T cells (P = 0.01). The changes in phosphocholine and glycerophosphocholine levels seen in cell extracts were also detectable in the 31P MR spectra of intact MDA-MB-231 cells following exposure to LY294002. When treated with another PI3K inhibitor, wortmannin, MDA-MB-231 cells also showed a significant decrease in phosphocholine content by ∼1.25-fold relative to the control (P < 0.05), whereas the levels of the remaining metabolites did not change significantly. Our results indicate that PI3K inhibition in human breast cancer cells by LY294002 and wortmannin is associated with a decrease in phosphocholine levels. [Mol Cancer Ther 2006;5(1):187–96]

Magnetic Resonance Spectroscopy Monitoring of Mitogen-Activated Protein Kinase Signaling Inhibition

Several mitogen-activated protein kinase (MAPK) signaling inhibitors are currently undergoing clinical trial as part of novel mechanism-based anticancer treatment strategies. This study was aimed at detecting biomarkers of MAPK signaling inhibition in human breast and colon carcinoma cells using magnetic resonance spectroscopy. We investigated the effect of the prototype MAPK kinase inhibitor U0126 on the (31)P-MR spectra of MDA-MB-231, MCF-7 and Hs578T breast, and HCT116 colon carcinoma cells. Treatment of MDA-MB-231 cells with 50 micromol/L U0126 for 2, 4, 8, 16, 24, 32, and 40 hours caused inhibition of extracellular signal-regulated kinases (ERK1/2) phosphorylation from 2 hours onwards. (31)P-MR spectra of extracted cells indicated that this was associated with a significant drop in phosphocholine levels to 78 +/- 8% at 8 hours, 74 +/- 8% at 16 hours, 66 +/- 7% at 24 hours, 71 +/- 10% at 32 hours, and 65 +/- 10% at 40 hours post-treatment. In contrast, the lower concentration of 10 micromol/L U0126 for 40 hours had no significant effect on either P-ERK1/ 2 or phosphocholine levels in MDA-MB-231 cells. Depletion of P-ERK1/2 in MCF-7 and Hs578T cells with 50 micromol/L U0126 also produced a drop in phosphocholine levels to 51 +/- 17% at 40 hours and 23 +/- 12% at 48 hours, respectively. Similarly, in HCT116 cells, inhibition with 30 micromol/L U0126 caused depletion of P-ERK1/2 and a decrease in phosphocholine levels to 80 +/- 9% at 16 hours and 61 +/- 4% at 24 hours post-treatment. The reduction in phosphocholine in MDA-MB-231 and HCT116 cells correlated positively with the drop in P-ERK1/2 levels. Our results show that MAPK signaling inhibition with U0126 is associated with a time-dependent decrease in cellular phosphocholine levels. Thus, phosphocholine has potential as a noninvasive pharmacodynamic marker for monitoring MAPK signaling blockade.

The Phosphoinositide 3-Kinase Inhibitor PI-103 Downregulates Choline Kinase α Leading to Phosphocholine and Total Choline Decrease Detected by Magnetic Resonance Spectroscopy

The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus ((31)P)- and proton ((1)H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103-induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase alpha (ChoK(alpha)), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r(2) = 0.9, P = 0.009) was found only between PC concentrations and ChoK(alpha) expression but not with FAS or pACL. This study identified inhibition of ChoK(alpha) as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of (1)H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with (31)P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors.

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.

Lactate and Choline Metabolites Detected In Vitro by Nuclear Magnetic Resonance Spectroscopy Are Potential Metabolic Biomarkers for PI3K Inhibition in Pediatric Glioblastoma

The phosphoinositide 3-kinase (PI3K) pathway is believed to be of key importance in pediatric glioblastoma. Novel inhibitors of the PI3K pathway are being developed and are entering clinical trials. Our aim is to identify potential non-invasive biomarkers of PI3K signaling pathway inhibition in pediatric glioblastoma using in vitro nuclear magnetic resonance (NMR) spectroscopy, to aid identification of target inhibition and therapeutic response in early phase clinical trials of PI3K inhibitors in childhood cancer. Treatment of SF188 and KNS42 human pediatric glioblastoma cell lines with the dual pan-Class I PI3K/mTOR inhibitor PI-103, inhibited the PI3K signaling pathway and resulted in a decrease in phosphocholine (PC), total choline (tCho) and lactate levels (p<0.02) as detected by phosphorus (31P)- and proton (1H)-NMR. Similar changes were also detected using the pan–Class I PI3K inhibitor GDC-0941 which lacks significant mTOR activity and is entering Phase II clinical trials. In contrast, the DNA damaging agent temozolomide (TMZ), which is used as current frontline therapy in the treatment of glioblastoma postoperatively (in combination with radiotherapy), increased PC, glycerophosphocholine (GPC) and tCho levels (p<0.04). PI-103-induced NMR changes were associated with alterations in protein expression levels of regulatory enzymes involved in glucose and choline metabolism including GLUT1, HK2, LDHA and CHKA. Our results show that by using NMR we can detect distinct biomarkers following PI3K pathway inhibition compared to treatment with the DNA-damaging anti-cancer agent TMZ. This is the first study reporting that lactate and choline metabolites are potential non-invasive biomarkers for monitoring response to PI3K pathway inhibitors in pediatric glioblastoma.

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.

In vitro nuclear magnetic resonance spectroscopy metabolic biomarkers for the combination of temozolomide with PI3K inhibition in paediatric glioblastoma cells

Recent experimental data showed that the PI3K pathway contributes to resistance to temozolomide (TMZ) in paediatric glioblastoma and that this effect is reversed by combination treatment of TMZ with a PI3K inhibitor. Our aim is to assess whether this combination results in metabolic changes that are detectable by nuclear magnetic resonance (NMR) spectroscopy, potentially providing metabolic biomarkers for PI3K inhibition and TMZ combination treatment. Using two genetically distinct paediatric glioblastoma cell lines, SF188 and KNS42, in vitro 1H-NMR analysis following treatment with the dual pan-Class I PI3K/mTOR inhibitor PI-103 resulted in a decrease in lactate and phosphocholine (PC) levels (P<0.02) relative to control. In contrast, treatment with TMZ caused an increase in glycerolphosphocholine (GPC) levels (P≤0.05). Combination of PI-103 with TMZ showed metabolic effects of both agents including a decrease in the levels of lactate and PC (P<0.02) while an increase in GPC (P<0.05). We also report a decrease in the protein expression levels of HK2, LDHA and CHKA providing likely mechanisms for the depletion of lactate and PC, respectively. Our results show that our in vitro NMR-detected changes in lactate and choline metabolites may have potential as non-invasive biomarkers for monitoring response to combination of PI3K/mTOR inhibitors with TMZ during clinical trials in children with glioblastoma, subject to further in vivo validation.

Abstract 5277: Non-invasive metabolic biomarkers of histone deacetylase inhibition in human colon cancer cells and tumors

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Histone deacetylase (HDAC) inhibitors are targeted anti-cancer agents currently approved for cutaneous T-cell lymphoma and in mid-late stage trials for other cancers (Ma et al, Drugs 2009). Developing non-invasive biomarkers of drug activity could provide a useful tool to aid clinical development. We have previously shown that the HDAC inhibitor LAQ824 increases phosphocholine (PC) levels in human colon cancer cells and tumors as detected by magnetic resonance spectroscopy (MRS), a non-invasive method for monitoring tissue metabolism (Chung et al, Neoplasia 2008). Here we sought to confirm this effect using a different chemotype probe (PXD101, belinostat) and investigate the mechanism(s) underlying it. Methods: Human HT29 colon cancer cells were treated for 24h with DMSO or 2 M PXD101 in DMEM ± 28 µM [1,2-13C]-choline. For in vivo evaluation, female nude mice bearing HT29 xenografts were treated i.p. with vehicle (n=6) or 60mg/kg PXD101 (n=6) for 3 days. In vitro metabolism was assessed using 31P or 13C MRS on cell extracts, while in vivo metabolism was evaluated using 1H and 31P MRS pre- (d0) and 3 days post-treatment, followed by analysis of excised tumor extracts. Western blotting for acetyl histone-3 was used to verify inhibitor action. Results: PXD101 treatment in HT29 cells induced histone-3 acetylation and decreased cell counts to 55±6% of controls (p=0.005). In vivo, PXD101 treatment resulted in tumor stasis (grew by 2% from d0) while the control tumors grew by 20% from d0. 31P MRS showed increased cellular PC content to 156±13% (p=0.04) post-PXD101 treatment. In vivo 1H and 31P MRS of tumors revealed rises in total choline/water (169±25%; p=0.02) and phosphomonoester (i.e. PC+phosphoethanolamine)/total phosphate ratios (p=0.01). Ex-vivo analysis attributed this effect to a rise in PC up to 1.4-fold (p=0.03) in the PXD101-treated tumors relative to controls. PC is formed mainly through phosphorylation of choline via choline kinase (de novo route) or hydrolysis of phosphatidylcholine via phospholipases. 13C MRS analysis of cells grown in the presence of the tracer [1,2-13C]-choline showed increased 13C- PC formation post-PXD101 treatment (180±19% of controls, p=0.02) consistent with increased de novo PC synthesis. Conclusions: Our data show that HDAC inhibition with PXD101 increases PC levels in human colon cancer cells and tumors thus confirming our previous findings with the different chemotype agent LAQ824 (Chung et al, Neoplasia 2008). Importantly, we show that this effect is driven by a rise in de novo PC synthesis. These data further support the role of PC as a potential non-invasive biomarker for monitoring the action of HDAC inhibitors. We acknowledge the support received for the CRUK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England). We also acknowledge NHS funding to the NIHR Biomedical Research Centre and NCI for providing PXD101. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5277. doi:10.1158/1538-7445.AM2011-5277

Abstract 3788: Autophagy induced by DCA, PI3K inhibition or starvation results in reduced lactate production measured in real-time by DNP 13C MRS

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL INTRODUCTION: Autophagy is a cellular degradation response to starvation or stress where cellular proteins, organelles and cytoplasm are engulfed, digested and recycled to sustain cellular metabolism [Mathew et al. Nat Rev Cancer (2007)]. Increased TCA cycle activity is predicted during autophagy, as the amino acids and fatty acids generated by the autophagic process are utilised in the TCA cycle. AIM: To investigate the effects of autophagy on TCA cycle activation in cells by dynamic nuclear polarisation (DNP) and 13C magnetic resonance spectroscopy (MRS) of pyruvate metabolism, in order to develop a non-invasive marker for autophagy. METHODS: Bax deficient colon carcinoma HCT116 cells (Baxko) were used to induce autophagy with 24 hrs of starvation or 10µM PI-103 (a PI3K inhibitor). These cells were also treated with 75mM dichloroacetate (DCA), a PDK inhibitor, to study the effect of TCA cycle activation [Bonnet et al. Cancer Cell (2007)] on lactate production. Electron microscopy, flow cytometry and western blots provided markers of autophagy and apoptosis. Real-time lactate production rate was monitored by [1-13C] pyruvate DNP assay and 13C MRS. Culture media and extracts from the treated and control cells were analysed by 1H MRS. Lactate dehydrogenase (LDH) expression and activity were examined. Surprisingly, autophagy was induced in HCT116 Baxko cells by DCA. This effect was further examined in HT29 (colon) and PC3 (prostate) cancer cells. RESULTS: Markers of autophagy by western blots (increased LC3II expression) and electron microscopy (presence of autophagosomes) confirmed the induction of autophagy in HCT116 Baxko cells by starvation, DCA or PI103 treatment and in DCA treated PC3 and HT29 cells. All treated groups exhibited minimal apoptosis or necrosis. Significant reductions in the rate of real-time lactate production by DNP 13C MRS were found in all treated groups. Decreases in steady-state lactate production were found in media from all treated groups. No change in LDH expression/activity or its co-factor NAD+ concentration was found in any treated group. DISCUSSION: Autophagy was induced in 3 cancer cell lines by 3 different methods. To our knowledge, this is the first study to show DCA induced autophagy in cancer cells. The observed reduction in real-time (and steady-state) lactate production was associated with autophagy. This finding together with unchanged cellular NAD+ and LDH could be due to more pyruvate being diverted to the TCA cycle; and/or LDH being sequestrated in autophagic vacuoles [Houri et al. Biochem J (1995)]. CONCLUSIONS: A reduction in lactate production measured by DNP 13C MRS, and unchanged NAD+, may provide a non-invasive means of assessing autophagy. We acknowledge CRUK, EPSRC, MRC and Department of Health for the Cancer Imaging Centre, NHS funding to the NIHR Biomedical Research Centre and Dr Paul Clarke for HCT116 Baxko cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3788. doi:10.1158/1538-7445.AM2011-3788