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Available online at www.sciencedirect.com
www.elsevier.com/locate/jprot
The phosphoinositide 3-kinase inhibitor LY294002, decreases aminoacyl-tRNA synthetases, chaperones and glycolytic enzymes in human HT-29 colorectal cancer cells Duthika M. Mallawaaratchy, Swetlana Mactier, Kimberley L. Kaufman, Katherine Blomfield, Richard I. Christopherson⁎ School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia
AR TIC LE I N FO
ABS TR ACT
Article history:
The proposed anticancer drug LY294002, inhibits phosphoinositide-3 kinase (PI3K) that ini-
Received 22 July 2011
tiates a signalling pathway often activated in colorectal cancer (CRC). The effects of
Accepted 25 November 2011
LY294002 (10 μM, 48 h) on the cytosolic, mitochondrial and nuclear proteomes of human
Available online 6 December 2011
HT-29 CRC cells have been determined using iTRAQ (isobaric tag for relative and absolute quantitation) and tandem mass spectrometry (MS/MS). Analysis of cells treated with
Keywords:
LY294002 identified 26 differentially abundant proteins that indicate several mechanisms
Colorectal cancer
of action. The majority of protein changes were directly or indirectly associated with Myc
LY294002
and TNF-α, previously implicated in CRC progression. LY294002 decreased the levels of 6
PI3 kinase
aminoacyl-tRNA synthetases (average 0.39-fold) required for protein translation, 5 glycolyt-
iTRAQ
ic enzymes (average 0.37-fold) required for ATP synthesis, and 3 chaperones required for
LC–MS/MS
protein folding. There was a 3.2-fold increase in lysozyme C involved in protein-glycoside hydrolysis. LY294002 increased cytosolic p53 with a concomitant decrease in nuclear p53, suggesting transfer of p53 to the cytosol where apoptosis might be initiated via the intrinsic mitochondrial pathway. Protein changes described here suggest that the anti-angiogenic effects of LY294002 may be related to p53; the mutational status of p53 in CRC may be an important determinant of the efficacy of PI3K inhibitors for treatment. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Colorectal cancer (CRC) is the second-most common cancer world-wide [1]. The risk of CRC increases with age, inherited mutations, obesity, physical inactivity, smoking and a low-fibre diet [2,3]. CRC develops as a multi-step process from adenoma to carcinoma that may take 15 years [4]. The first stage may be associated
with loss of the APC gene, resulting in hyperactive Wnt signalling and proliferation of cells beyond the intestinal crypts. This is followed by mutations in K-Ras and phosphoinositide-3 kinase (PI3K) catalytic subunit alpha, or PTEN (phosphatase and tensin homologue deleted on chromosome 10) [5]. Loss of the tumour suppressor protein p53, deregulates cell proliferation, blocks stress-induced apoptosis and promotes progression from
Abbreviations: CRC, colorectal cancer; DMSO, dimethyl sulfoxide; ECL, enhanced chemiluminescence; ESI, electrospray ionization; FCS, fetal calf serum; HRP, horse-radish peroxidase; IC50, half maximal inhibitory concentration; IEF, isoelectric focusing; IPG, immobilized pH gradient; LC–MS/MS, liquid chromatography–mass spectrometry; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1 benzopyran-4-one; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PMF, peptide mass finger-printing; PTEN, phosphatase and tensin homologue deleted on chromosome 10; SCX, strong cation exchange; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TEAB, triethylammonium bicarbonate. ⁎ Corresponding author. Tel.: +61 2 9351 6031; fax: + 61 2 9351 4726. E-mail address:
[email protected] (R.I. Christopherson). 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.11.032
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adenoma to carcinoma [6,7]. Early detection and surgical removal of primary tumours is still the most successful treatment [8]. If metastasis has occurred, a combination of chemotherapy (such as 5fluorouracil or oxaliplatin) and radiation is used. However, these regimes have a poor 5-year survival rate of ~10% [8]. Approximately 60% of sporadic CRC cases have high levels of activated Akt [9]. There is no mutation common to most CRCs, but of the ~90 somatic mutations in a tumour, at least one usually affects the PI3K pathway [6,10–13]. This high mutation frequency, taken with the range of oncogenic processes controlled by PI3K signalling (angiogenesis, metabolism, cell proliferation, differentiation and apoptosis), makes PI3K an ideal target for anticancer drugs. CRC cells that are dependent on this pathway to maintain the cancerous phenotype will be more sensitive to inhibition of PI3K signalling than normal cells. PI3K inhibitors would show selective toxicity for these CRCs compared with normal tissues [14–18]. PI3Ks are divided into sub-families that regulate a number of processes from cell proliferation to vesicle trafficking. Class IA PI3K catalyzes formation of phosphatidylinositol 3,4,5trisphosphate (PIP3) [19], is often mutated in CRC [12,13], and binds to a p85 adapter protein and K-Ras [19,20]. They may be activated by receptor tyrosine kinases (RTK) via p85, or directly by KRas. In the presence of PIP3, Akt at the plasma membrane is phosphorylated at Thr308 and Ser473, which in turn phosphorylates a number of downstream effectors including mTOR, MDM2, NFκB, and the BAD/BCL-XL complex [21,22]. The phosphorylation state of Akt is often used as a measure of PI3K signalling[23–26]; overexpression of p-Akt is found in late-stage and metastatic CRC [27]. LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1 benzopyran-4one), is a synthetic, first-generation pan-PI3K inhibitor based on the structure of quercetin, a plant-derived PI3K inhibitor [28]. LY294002 inhibits PI3K by binding reversibly to the ATP site of PI3K [9,28–30]. Low solubility, short half-life and low potency have limited clinical use of LY294002 [31], however a LY294002 derivative, SF1126, is currently undergoing Phase I clinical trials [17,32]. The chemical structure of SF1126 is identical to LY294002 except for addition of an RDGS (ArgGly-Asp-Ser) integrin-binding element that directs the drug to the tumour vasculature and increases solubility [33]. The RDGS peptide is removed inside the cell, and LY294002 then inhibits PI3K [31]. LY294002 and SF1126 show anti-angiogenic and anti-tumour effects, inducing activation of p53 and cell cycle arrest [22]. LY294002 inhibits growth and induces apoptosis in CRC, T-cell leukaemia, pancreatic cancer, androgensensitive prostate cancer and malignant glioma [25,34,35]. The effects of PI3K inhibition on cellular functions must be evaluated to assess the selective toxicity of LY294002, the effects on the cellular proteome are largely unknown. Proteomic analysis by iTRAQ™ peptide labelling and tandem mass spectrometry (MS/MS) enables identification and quantitation of proteins that are differentially abundant in drug-treated cells [36,37]. Inhibition of PI3K may induce compensatory activation of alternate kinase pathways that would be synergistic drug targets. Sub-cellular fractionation has been used to reduce the complexity of cell extracts, enabling detection of low abundance proteins and information on the cellular localization of differentially-abundant proteins. Cell lines capable of being maintained in continuous culture must acquire additional mutations, compared with the original cancer and may not closely resemble the original cancer. Cell
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lines are different from primary cancer cells, but may have similar morphology, immunophenotype, karyotype, cytogenetics and molecular characteristics, providing unique in vitro models for studying cellular and molecular events in CRC. The HT-29 cell line was derived from a colonic adenocarcinoma of a 44 yearold female patient [38]. HT-29 cells have an activating mutation in PIK3CA, the catalytic subunit of PI3K, that leads to increased basal PI3K signalling. HT-29 cells are sensitive to PI3K inhibition by LY294002 [39]. The HT-29 cell line also has mutations in APC, B-Raf, SMAD4 and p53. The Arg273His mutation in p53 seen in HT-29 is a missense mutation that lowers the DNA binding affinity and reduces transcriptional activity of p53 compared to wild type [40,41]. This may lead to greater sensitivity to drugs as reported and seen by Hart et al. where there was a greater sensitivity to irinotecan, in the mutated p53 cell line HT-29 compared to a modified wt p53 transfected subclone HT-29A4. In this study, the effects of LY294002 on the cytosolic, mitochondrial and nuclear proteomes of HT-29 cells have been determined. The results obtained provide further insight regarding the mechanisms of action of this PI3K inhibitor.
2.
Materials and methods
2.1.
Growth of HT-29 cells and drug treatment
Human HT-29 CRC cells (American Type Culture Collection, Manassas, VA, USA) were grown as an adherent monolayer in DMEM containing 15 mM HEPES buffer, supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 14.2 mM NaHCO3, pH 7.2 at 37 °C. Cell growth was determined using the Sulforhodamine B (SRB) assay described by Voigt et al. [42]. Briefly, adherent cells were trypsinised and suspended in growth medium. Cells were seeded into a 96-well plate at ~3000 cells/well and incubated overnight at 37 °C. The cultures were then treated with DMSO ± 10 μM LY294002 and samples were taken up to 120 h. Growth was halted by addition of 2.5% (w/v) TCA, the cells were maintained at 4 °C for 1 h, stained with SRB solution (0.4% (w/v) SRB in 1% (v/v) acetic acid) at 4 °C for 30 min, washed with 1% (v/v) acetic acid and air-dried. The cells were then de-stained with 10 mM Tris base (pH 10.0) and the absorbance was read at 560 nm with a 96-well plate reader (Original Multiskan EX, Thermo Electron Corporation, Shanghai, China). HT-29 cells were grown in exponential phase in triplicate cultures and treated with 10 μM LY294002 in DMSO for 48 h at 37 °C (IC50 concentration). Control cultures were treated with equivalent volumes of DMSO. The inhibitory effects of LY294002 on the PI3K pathway were assessed by Western blotting for phosphorylated Akt using a phospho-Akt (Ser 473) (pAkt) antibody (1:1000) on extracts of untreated and LY294002-treated cells (10 μM, 48 h). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal loading control (see Western blotting below).
2.2. Sub-cellular fractionation of HT-29 cells and protein extraction Cytosolic, mitochondrial and nuclear fractions were prepared by differential centrifugation as described by Mactier et al. [43]. Briefly, HT-29 cells (5×108, three biological replicates) were trypsinised
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(0.25% (w/v), 5 min, 37 °C, GIBCO, Grand Island, NY, USA), centrifuged (400 g, 5 min, room temperature) and incubated in lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1% (v/ v) NP-40, 0.5 mM phenylmethanesulfonylfluoride, PMSF) on ice for 5 min before homogenisation with 8 strokes of a Dounce homogenizer. The resulting homogenate was centrifuged (800 g, 10 min, 4 °C) to remove crude nuclei and cell debris. The supernatant was centrifuged (15,000 g, 25 min, 4 °C) to isolate crude mitochondria, followed by centrifugation (100,000 g, 60 min, 4 °C) to isolate the cytosolic fraction. Crude mitochondria were resuspended in isotonic buffer (10 mM Tris–HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA), purified on a 1/1.5 M sucrose step gradient (10 mM Tris–HCl pH 7.4, 1 M or 1.5 M sucrose, and 1 mM EDTA) and centrifuged with the brake off (87,000 g, 2 h, 4 °C). Purified mitochondria were obtained from the 1/1.5 M sucrose interface, rinsed in an equal volume of buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 M EDTA) and centrifuged (15,000 g, 20 min, 4 °C) to pellet mitochondria. The crude nuclear fraction was resuspended in isotonic buffer and layered onto a 1.3/2.3 M sucrose step gradient and centrifuged (5000 g, 45 min, 4 °C). The purified nuclear pellet was rinsed in isotonic buffer and centrifuged (1000 g, 5 min, 4 °C). Protein extracts were processed using the ReadyPrep™ 2-D Cleanup Kit from Bio-Rad (Hercules, CA, USA) according to the manufacturer's protocol. Protein concentrations were determined using the 2D Quant Kit (GE Healthcare, Piscataway, NJ, USA) following the manufacturer's instructions. The purity of the sub-cellular fractions was confirmed by Western blotting for marker proteins (see below).
2.3.
iTRAQ labelling
Sub-cellular proteomes of HT-29 cells, control (n = 3) and LY294002-treated (n = 3), were digested with trypsin and labelled with iTRAQ tags according to the manufacturer's protocol (AB SCIEX, Foster City, CA, USA). Proteins (20 μg) were diluted in 0.5 M triethylammonium bicarbonate (TEAB), pH 8.5 and 0.1% (w/v) SDS, and reduced with 50 mM tris(2-carboxyethyl) phosphine (TCEP) for 1 h at 60 °C. To block cysteine residues, samples were incubated at room temperature for 10 min with 200 mM methyl methane thiosulfonate (MMTS). Sequencing grade modified trypsin (Promega, Madison, WI, USA) was added at a ratio of 1:25 (trypsin:protein) and incubated at 37 °C for 2 h. Protein digestion was completed with a second addition of trypsin (1:25) with incubation at 37 °C for 16 h. Samples were dried in a Vacuum Centrifugal Concentrator 5301 (Eppendorf, Hamburg, Germany) and then resuspended in 7 μL 1 M TEAB, pH 8.5. Samples were labelled with isotopic tags as follows: Control 1, 113; Control 2, 114; Control 3, 115; LY294002 1, 116; LY294002 2, 117 and LY294002 3, 118. The iTRAQ reagents were dissolved in isopropanol, 20 μL of this iTRAQ mix was added to each peptide sample to give a final isopropanol concentration of 65% (v/v), and incubated at room temperature for 2 h. After labelling, samples for each subcellular fraction were combined prior to analysis.
2.4. Strong cation-exchange chromatography and reverse-phase LC–MS/MS Prior to MS analysis, peptides from each sub-cellular fraction were separated using a strong cation-exchange column
(ZORBAX Bio-SCX series II, 3.5 mm, 50 × 0.8 mm, Agilent, Palo Alto, CA, USA) on an 1100 HPLC system (Agilent, Palo Alto, CA, USA) in off-line mode. The peptides (30 μg) were loaded onto the column in SCX buffer A (25% (v/v) ACN, 0.05% (v/v) formic acid, pH 2.5) and eluted with a gradient from 0 to 20% SCX buffer B (25% (v/v) ACN, 0.5 M ammonium formate, 2% (v/v) formic acid, pH 2.5) in 42 min (14 min, 20–100% B; 5 min, 100% B). Thirty fractions were collected at 2-min intervals using a Probot micro-fraction collector (Dionex/LC Packings, Sunnyvale, CA, USA). The SCX fractions were analysed using an Agilent 1100 HPLC system interfaced with a QSTAR Elite mass spectrometer (AB SCIEX, Foster City, CA, USA). The SCX fractions were diluted with solvent A (0.1% (v/v) formic acid) and loaded onto a C18 trap column (ZORBAX 300SB-C18, 300 μm × 5 mm, 5 μm, Aligent) at 10 μL/min and washed for 7 min before switching the trap column in line with the C18 separation nano-column (ZORBAX 300SB C18, 3.5 μm, 150 × 0.1 mm, Agilent). The peptides were eluted directly into the ionization source of the mass spectrometer at 0.6 μL/min with the following gradient: 0 min, 5% solvent B (0.1% (v/v) formic acid in ACN); 8 min, 5% B; 10 min, 15% B; 90 min, 30% B; 105 min, 60% B; 115 min, 5% B; 120 min, 5% B. The nano-LC-ESI-MS/MS system was set for data acquisition in the positive ion mode, with a selected range of 350–1750 m/z. Peptides with +2 to +4 charge states were selected for tandem mass spectrometry, and the time of summation of MS/MS events was 2 s. The three most abundantly charged peptides above a count threshold >30 were selected for MS/MS and dynamically excluded for 30 s with (50 ppm mass tolerance). The instrument ran in information dependent acquisition (IDA) mode using Analyst QS 2.0 software (AB SCIEX Inc., Foster City, CA, USA). Automatic collision energy and MS/MS accumulation modes were used in the advanced IDA settings. A modified Enhance All mode Q2 transition setting was used favouring low mass ions so that the iTRAQ reporter ions (113–119, 121) intensities were enhanced for quantification.
2.5.
Data analysis
iTRAQ MS/MS data were analysed using ProteinPilot 3.0 software (AB SCIEX), which uses the Paragon™ algorithm to perform protein identification. All MS/MS spectra were searched against a combined Swiss-Prot protein database, version uni_sprot_20070123. Parameters set in ProteinPilot 3.0 included (i) sample type, iTRAQ 8-Plex, (ii) cysteine alkylation, MMTS, (iii) digestion, trypsin allowing two missed cleavages, (iv) species, Homo sapiens, (v) instrument, QSTAR Elite and (vi) special factors, urea denaturation. The following processing options were used: quantitative, bias correction, background correction, biological modifications and thorough identification search. A concatenated target-decoy database search strategy was also employed to estimate the rate of false positives. Only proteins identified with at least 95% confidence and unused ProtScore of >1.3 were reported. Relative peptide abundances were determined using the MS/MS scans of iTRAQ-labelled peptides, where the ratios of peak areas of the iTRAQ reporter ions reflect the relative abundances of the peptides and therefore, the proteins in the samples. The results obtained using the ProteinPilot 3.0 software were exported to Microsoft Excel® for further analysis. A two-
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tailed Student's t-test was performed on the data with equal variance between control and LY294002-treated samples. The average ratios of control to LY294002 samples were calculated and used to determine the fold-changes with treatment. Protein level changes less than 0.75-fold or greater 1.25-fold (p< 0.05) were considered biologically significant. Proteins identified following iTRAQ analysis were classified into biological pathways using Ingenuity Pathway Analysis software (IPA; Ingenuity® Systems, Redwood City, CA, USA; www.ingenuity. com). This software calculates the probability that the genes associated with a dataset (identified proteins) are involved in particular biological functions and canonical pathways. Significance values were calculated using the right-tailed Fisher's Exact Test by comparing the number of proteins that occur in a given pathway relative to the total number of occurrences of those proteins in all functional annotations stored in the Ingenuity Pathways Knowledge Base.
2.6.
Western blotting
Proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane at 400 mA for 1 h using a Criterion™ Blotter (BioRad, Hercules, CA, USA). The membrane was incubated overnight at 4 °C with primary antibody (Phospho-Akt (Ser 473) Phospho-Akt (Thr 308)); Akt; HSP90 (C45G5); PKM2; (Cell Signalling Technology, Arundel, Queensland, Australia); TCP1 delta; seryl-tRNA synthetase; (Abcam, Waterloo, NSW, Australia); Lysozyme C (W-20); peIF2α; SDHA; PU.1; p53 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and glyceraldehyde 3-phosphate dehydrogenase GAPDH; (Millipore, North Ryde, Australia). The membrane was then incubated with secondary antibody conjugated to horse-radish peroxidase (HRP) for 2 h at room temperature: donkey, anti-rabbit, (Abcam, Waterloo, NSW, Australia); bovine, anti-goat and goat, anti-mouse (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The bands were visualised with ECL detection reagent and imaged using chemiluminescence film (GE Healthcare, Piscataway, NJ, USA). For proteins/antibodies with weak signals, Signal Boost Immunoreaction Enhancer Kit (Calbiochem, San Diego, CA, USA) and RapidStep ECL Reagent (Calbiochem, San Diego, CA, USA) were used to visualise the protein bands. Changes in proteins of interest are shown as a ratio with the density of the house-keeping protein, GAPDH (except for nuclear fractions where proteins are expressed as a ratio with PU.1). Student's t-tests were used to determine the significance of changes by comparing the LY294002-treated cells with controls. Bands were quantified using ImageQuantTL density analysis software (GE Healthcare).
3.
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LY294002 for 48 h. The marker proteins glyceraldehyde 3phosphate dehydrogenase (GAPDH, cytosol), succinate dehydrogenase A (SDHA, mitochondria) and PU.1 (nucleus) were detected in whole cell extracts and the corresponding subcellular fractions (see Fig. S2 in Supplementary material). To assess the inhibitory effect of LY294002 on the PI3K pathway, Akt phosphorylation was measured by Western blotting. There were significant decreases in 60-kDa pAkt(S473) and pAkt(T308) compared with total Akt following LY294002 treatment (p< 0.05, Fig. 1), indicating inhibition of the PI3K pathway.
3.2. Identification of differentially abundant proteins using iTRAQ and LC–MS/MS
Differentially abundant proteins were identified in the subcellular fractions of control and LY294002-treated HT-29 cells. Using a ProtScore >1.3 (95% confidence, p < 0.05) as a cut-off, a total of 2481 proteins were identified, including 760 cytosolic, 719 mitochondrial and 1002 nuclear proteins. Of these, 26 proteins became differentially abundant following LY294002 treatment (p< 0.05; Table 1); 21 proteins decreased by at least 0.75fold and 5 proteins increased by 1.5-fold or more (Table 1). The majority of differentially abundant proteins were cytosolic (76%), followed by mitochondrial (20%) and nuclear (4%). The IPA software clustered proteins altered by LY294002 treatment according to their biological functions (see Fig. S3 in Supplementary material). Functions significantly affected following PI3K inhibition (p < 0.05) included carbohydrate metabolism, post-translational modification, protein folding, cancer and gastrointestinal disease. The top-scoring networks for proteins differentially abundant in HT-29 cells treated with LY294002 were cancer, gastrointestinal disease, and post-translational modification (score= 66). A protein network describing the direct and indirect relationships of 13 of the 26 proteins identified was generated (see Fig. S4 in Supplementary material).
Results
3.1. Cellular growth, sub-cellular markers and Akt phosphorylation
LY294002 (10 μM) reduced the growth of HT-29 cells measured by the SRB assay to 59% (p < 0.0001) after 48 h and to 65% (p < 0.0001) after 120 h (see Fig. S1 in Supplementary material). For proteomic analysis, cells were incubated with 10 μM
Fig. 1 – Effect of LY294002 on pAkt in HT-29 cells. (A) Whole cell extracts ± LY294002 (10 μM, 48 h) were prepared and 30 μg protein from control (C) and LY294002-treated cells (Ly) was separated by 10% SDS-PAGE for Western blotting with antibodies for pAkt(S273), pAkt(T308), Akt and GAPDH (loading control). (B) The intensity of GAPDH was used to normalise values for control and treated cells. (*), p < 0.05; error bars show mean ± standard deviation. The figure is representative of three independent experiments.
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Table 1 – Differentially abundant proteins identified by iTRAQ analysis of HT-29 cells treated with LY249002 (10 μM, 48 h; p < 0.05). The function and sub-cellular location of identified proteins are indicated. Accession number a
Name
Proteins down-regulated by LY294002 Q5T5C7 Seryl-tRNA synthetase B4DEG8
P14618 P50991 P04075 Q9Y617 P41250
cDNA FLJ53464, highly similar to Threonyl-tRNA synthetase, cytoplasmic Pyruvate kinase isozymes M1/M2 T-complex protein 1 subunit delta Fructose-bisphosphate aldolase A Phosphoserine aminotransferase Glycyl-tRNA synthetase
P00558 P13667 O43707
Phosphoglycerate kinase 1 Protein disulfide-isomerase A4 Alpha-actinin-4
P23381
Tryptophanyl-tRNA synthetase
P49588
Alanyl-tRNA synthetase
P60174 P54577
Triosephosphate isomerase Tyrosyl-tRNA synthetase
P08238 P60981
Heat shock protein HSP 90-beta Destrin
IPI00887 678.1 P07237 P78347 P06733 P07900
LOC654188 similar to peptidylprolyl isomerase A-like Protein disulfide-isomerase General transcription factor II-I Alpha-enolase Heat shock protein HSP 90-alpha
Proteins up-regulated by LY294002 Q9NU36 Novel protein similar to small nuclear ribonucleoprotein polypeptide A' SNRPA1 Q8NF14 Protein FAM10A5 O14745 Ezrin-radixin-moesin-binding phosphoprotein 50 P30533 Alpha-2-macroglobulin receptorassociated protein P61626 Lysozyme C a b c d e f
Major function b
Gene
Subcellular location c
Peptides Unique pRatio (95%) d peptides e value Ly/ Cf
Aminoacyl-tRNA synthetase ligase Aminoacyl-tRNA synthetase ligase
SARS
Cytosol
4
5
0.01
0.25
TARS
Cytosol
3
4
0.01
0.27
Glycolysis Chaperone Glycolysis Transaminase Aminoacyl-tRNA synthetase ligase Glycolysis Chaperone Structural constituent of cytoskeleton Aminoacyl-tRNA synthetase ligase Aminoacyl-tRNA synthetase ligase Glycolysis Aminoacyl-tRNA synthetase ligase Chaperone Actin polymerization or depolymerization Isomerase
PKM2 CCT4 ALDOA PSAT1 GARS
Cytosol Cytosol Cytosol Cytosol Cytosol
61 5 45 5 7
20 3 15 3 4
0.05 0.00 0.01 0.04 0.04
0.28 0.32 0.33 0.33 0.35
PGK1 PDIA4 ACTN4
Cytosol Cytosol Cytosol
77 33 79
20 20 20
0.02 0.03 0.04
0.36 0.43 0.45
WARS
Cytosol
4
5
0.04
0.47
AARS
Cytosol
8
7
0.05
0.49
TPI1 YARS
Cytosol Cytosol
49 9
5 6
0.01 0.05
0.51 0.53
HSP90AB1 Mitochondria 11 DSTN Cytosol 9
6 7
0.02 0.02
0.58 0.61
–
Mitochondria 10
9
0.01
0.68
Isomerase Transcription factor Glycolysis Chaperone
P4HB GTF21 ENO1 HSP90AA1
Mitochondria Nucleus Mitochondria Mitochondria
7 19 4 6
6 20 3 4
0.01 0.05 0.04 0.01
0.68 0.70 0.71 0.72
Ribonucleoprotein
–
Nucleus
11
9
0.05
Ly/C 1.53
Binding protein Receptor binding
FLJ00388 SLCPA3R1
Cytosol Cytosol
6 8
5 7
0.01 0.01
1.58 1.89
Chaperone
LRPAP1
Cytosol
3
3
0.01
2.63
Glycosyl hydrolase
LYZ
Cytosol
1
3
0.04
3.18
Accession numbers of proteins were derived from the Swiss-Prot database. Protein function and biological process were assigned in accordance with the Human Protein Reference Database (http://www.hprd.org/). Experimental sub-cellular location of proteins. Peptides with over 95% confidence. Number of unique peptides used to identify a protein. Average abundance of protein in LY294002 divided by protein in control.
Western blotting confirmed changes in 5 differentially abundant proteins after LY294002 treatment: HSP90, CCT4, PKM2, seryl-tRNA synthetase and lysozyme C (Fig. 2), selected for functional significance, antibody availability and high fold-changes. Several differentially abundant proteins were involved in protein synthesis (e.g., 6 aminoacyl-tRNA synthetases, CCT4, PDIA4 and HSP90). To examine the overall effect
of LY294002 on protein synthesis, we measured the phosphorylation state of the α-subunit of eukaryotic initiation factor 2 (eIF2α), a translation factor that controls protein synthesis. No increase in eIF2α phosphorylation was detected after LY294002, indicating that global protein synthesis is unaffected (Fig. 3). Since tryptophanyl-tRNA synthetase (WRS) interacts with p53, the cytosolic, nuclear and total cellular levels of p53
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Fig. 3 – Effect of LY294002 on peIF2α. (A) Whole cell extracts ± LY294002 (10 μM, 48 h) were prepared and 30 μg proteins from control (C) and LY294002-treated cells (Ly) were separated by 10% SDS-PAGE for Western blotting with antibodies for peIF2α and GAPDH (loading control). (B) The intensity of GAPDH was used to normalise values for control and treated cells. (*), p < 0.05; peIF2α, phospho eukaryotic initiation factor 2α. The figure is representative of three independent experiments.
4.
Discussion
Mutations affecting the PI3K/Akt signalling pathway may promote uncontrolled growth in cancer cells [20]; constitutive activation of Akt promotes cell survival and resistance to drugs and radiotherapy [44]. LY294002 has been shown to halt cancer growth by inhibiting PI3K signalling [45,46], but the exact mechanisms of action of this drug are unknown. We have
Fig. 2 – Validation by Western blotting of differentially abundant proteins in HT-29 cells treated with LY294002 (10 μM, 48 h). Proteins (15 μg of extract) were separated on 10% SDS-PAGE (15% SDS PAGE for LYZ) for Western blotting. (A) HSP90 (WCL); (B) PKM2 (WCL); (C) SRS (WCL); (D) CCT4 (WCL, cyto); (E) LYZ (cyto). The intensity of GAPDH was used to normalize values for control and treated cells. The figure is representative of three independent experiments. (*), error bars show mean± standard deviation, p < 0.05; WCL, whole cell lysate; cyto, cytosolic fraction; HSP90, heat shock protein 90; CCT4, T-complex protein 1δ; LYZ, lysozyme C; PKM2, pyruvate kinase, M1/M2; SRS, seryl-aminoacyl tRNA synthetase.
were assessed by Western blotting following LY294002. An increase in cytosolic p53 was observed, while p53 decreased in the nucleus and in whole cell lysates consistent with LY294002 inducing transfer of p53 to the cytosol (p < 0.05; Fig. 4).
Fig. 4 – Effect of LY294002 on p53. (A) Whole cell lysate, (B) cytosolic fraction (Cyto), and (C) nuclear fraction ± LY294002 (10 μM, 48 h), were prepared and 10 μg proteins from control (C) and LY294002-treated cells (Ly) were separated by 10% SDS-PAGE for Western blotting with antibodies for p53, GAPDH and PU.1 (loading controls). The intensity of GAPDH or PU.1 was used to normalize values for the control and treated cells (*), p < 0.05; error bars indicate mean ± SD. The figure is representative of three independent experiments.
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identified a number of proteins that change in abundance in HT-29 cells treated with LY294002 (Table 1), consistent with multiple mechanisms of action. The IPA software linked a network of proteins to Myc, a transcription factor that regulates cell growth and proliferation (see Fig. S4 in Supplementary material) [47]. Growth factors (e.g., VEGF) and their receptors are over-expressed in CRC [48–53], these growth factors promote tumour progression by up-regulating transcription factors, including c-Myc, through activating Akt/PI3K [54,55]. Inhibition of the PI3K pathway could affect multiple proteins regulated by Myc, including some identified here (Table 1). This network also implicates tumour necrosis factor (TNF) through direct and indirect interactions with proteins identified here (Table 1). TNF-α, produced by macrophages and tumour cells, binds to high-affinity cell surface receptors on CRC cells and induces multiple effects including apoptosis [56]. Over-expression of TNF-α is associated with CRC progression and reduced survival [57]. Inhibition of the PI3K signalling pathway by LY294002 has been linked to reduced TNF-α activity in HT-29 cells [58,59], consistent with the proteins identified here whose expression is modulated by TNF-α.
4.1.
4.3. Decreased protein chaperones and increased lysozyme C
Decreased aminoacyl-tRNA synthetases
LY294002 induced decreases in 6 class II aminoacyl-tRNA synthetases (aaRS; seryl-, threonyl-, glycyl-, tryptophanyl-, alanyl- and tyrosyl- tRNA synthetases; by 0.25-fold, 0.27-fold, 0.35-fold, 0.47-fold, 0.49-fold and 0.53-fold, respectively) in the cytosol of HT-29 cells (see Fig. S5 in Supplementary material), suggesting a reduction in protein biosynthesis and cell proliferation [60]. Levels of these aaRS may also be regulated by growth rate, protein synthesis and availability of individual amino acids [61]. To determine whether protein synthesis was impaired by LY294002, phosphorylation of eIF2α was determined by Western blotting. Phosphorylation of eIF2α inhibits translation decreasing protein synthesis [62], but no change was observed (Fig. 3). This suggests that protein synthesis was not inhibited by an eIF2α dependent mechanism. Further studies such as metabolic labelling with radioactive amino acids would clarify whether protein synthesis is inhibited by an aaRS dependent mechanism. The aaRS may have other regulatory roles not directly related to protein synthesis [63,64].
4.2.
consistent with transfer of p53 from the nucleus to the cytosol in HT-29 cells treated with LY294002 (p < 0.05; Fig. 4). The decrease in WRS induced by LY294002 could be linked to the increase of p53 (Fig. 4), although the Arg273His mutation present in HT-29 cells would lead to loss of function. The regulation of p53 nucleo-cytosolic shuttling is poorly understood, several proteins have been implicated, one being Mdm2 [70]. Inhibition of the PI3K/Akt pathway by LY294002 prevents nuclear translocation of Mdm2 leading to increased p53 in the cytosol [71]. LY294002 may modulate tumour-induced angiogenesis by regulating p53 transcription [22]. In response to stress, p53 accumulates in the nucleus to exert its effects [70]. Localization of p53 in the cytosol leads to lack of response to DNA damage in many cancers (including CRC), tumour metastasis and poor long-term survival [72–75]. The specific mechanisms of cytoplasmic tethering and accumulation of p53 are poorly understood, but, the mutational status of p53 in CRC may be an important determinant in selection of PI3K inhibitors for cancer treatment [76]. Further studies are required to confirm the effects of LY294002 on p53 in HT-29 cells.
Increased cytosolic p53
Interestingly, tryptophanyl-tRNA synthetase (WRS) is differentially expressed in CRC and may have prognostic significance for patients [65]. WRS binds to the vascular endothelial growth factor (VEGF) receptor with vascular endothelial-cadherin inhibiting the PI3K/Akt pathway, and arresting new blood vessel formation [66,67]. However, endothelial cells are not present in HT-29 cultures. Mutation of p53 in CRC in vivo is associated with increased VEGF expression and vessel counts [68]. In CRC cell lines treated with LY294002, increased p53 down-regulated WRS mRNA [69]. LY294002 induces expression of wild-type p53 [22] and mutant p53 (Arg273His) in HT-29 cells. Data from Western blotting are
Heat shock protein 90 (α/β) (HSP90) family proteins decreased (0.58-fold, Table 1) in the mitochondrial fraction of HT-29 cells treated with LY294002. Although typically located in the cytoplasm and endoplasmic reticulum, HSP90 has been isolated from the mitochondria of tumour cells [77]. HSP90 is a chaperone that assists with protein folding including signalling molecules like PI3K/Akt [20,78]. Cancer cells may be ‘addicted’ to HSP90 as it is a chaperone for many oncoproteins, a decrease in HSP90 results in degradation of client proteins via the ubiquitin-dependent proteasome pathway [79]. HT-29 cells express mutant BRAF (Val600Glu), a client protein of HSP90. Oncogenic proteins such as mutant BRAF can lead to constitutive activation of MAPK [80]. The signal transduction pathways involving MAPK and PI3K are activated by common growth factors, each pathway is necessary, yet insufficient alone, to stimulate proliferation [81]. Decreased HSP90 may induce apoptosis, possibly by overloading the protein degradation machinery [82,83]. T-complex protein 1 δ-subunit (CCT4, 0.32-fold, Table 1) decreases in the cytosol following LY294002 treatment. Cytosolic CCT4 is abundant in early S-phase, assisting the folding of actin, tubulin and other cytoskeletal proteins. A decrease in CCT therefore indicates that a cell is not actively dividing [84]. Similar to HSP90, CCT client proteins misfold when CCT is less abundant [84], down-regulation of CCT with siRNA decreases cell proliferation and apoptosis [85]. Protein disulphide isomerase (PDI) catalyses the re-arrangement of disulphide bonds during protein biosynthesis. Mitochondrial PDI-A4 plays an important role in cell invasion and protein folding, essential to cell survival under stress conditions [86,87]. Inhibition of PDI activity was found to increase apoptosis of melanoma cells [86,88]. In the present study, cytosolic PDI-A4 decreased (0.43-fold, Table 1) after LY294002 treatment, suggesting PI3K inhibition may promote apoptosis in HT-29 cells. Lysozyme C, a glycosyl hydrolase, increases
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3.18-fold following LY294002 treatment (Table 1) suggesting enhanced protein degradation in HT-29 cells. Taken together, the changes in PDI, CCT4, lysozyme C and HSP90 induced by LY294002 are likely to result in apoptosis and decreased growth of HT-29 cells.
4.4.
Decreased glycolytic enzymes
Inhibition of the PI3K pathway slows glycolysis; PI3K is a signal mediator downstream of the insulin receptor that activates translocation of the glucose transporter GLUT4, to the cell surface. Patients taking PI3K inhibitors might therefore have reduced uptake of glucose. Consistent with this effect, we identified 5 glycolytic enzymes whose levels are reduced by LY294002 (pyruvate kinase M1/M2, PKM2, 0.28-fold; fructose-bisphosphate aldolase A, 0.33-fold; phosphoglycerate kinase 1, 0.36-fold; triosephosphate isomerase, 0.51-fold; alpha enolase, 0.71-fold, Table 1). PKM2 is the rate-limiting enzyme of glycolysis, translocation of PKM2 to the nucleus can induce apoptosis [89], but an increase in nuclear PKM2 was not detected. The observed depletion of 5 glycolytic enzymes may inhibit ATP synthesis and cell proliferation. Many cancers are dependent upon substrate level phosphorylation via glycolysis to make ATP, called the Warburg effect. The Warburg effect refers to the high rate of aerobic glycolysis found in many cancer cells necessary to maintain the high energy requirements for growth and proliferation [90]. Aerobic glycolysis is much less efficient than oxidative phosphorylation in producing ATP, the advantage of cancer cells using substrate-level phosphorylation is unclear [91]. The activation of PI3K and the Warburg effect in cancer cells highlight the connection between signalling pathways that drive cell growth and metabolism [92]. Indeed, PI3K inhibitors have been shown to reduce lactate production, reversing the Warburg effect in cancer cells [92].
5.
Conclusions
LY294002 induces multiple proteomic effects in the sub-cellular compartments of human CRC cells. LY294002 reduces levels of 6 aminoacyl tRNA synthetases, 3 protein chaperones and 5 enzymes of glycolysis. Approximately 60% of sporadic CRC cases have high levels of activated pAkt [9]. Data presented here show that LY294002 inhibits PI3K in HT-29 cells indicated by reduction in the levels of activated pAkt. Accumulation of cytosolic p53, perhaps derived from nuclear p53, may induce apoptosis via the intrinsic pathway involving mitochondria. Major decreases in aminoacyl tRNA synthetases are likely to inhibit synthesis of some proteins, but no decrease in global protein synthesis was observed, as indicated by unchanged levels of phosphorylated eIF2. Decreases in 3 chaperone proteins are likely to decrease the growth of HT-29 cells. The depletion of 5 glycolytic enzymes may inhibit growth if the Warburg effect is important in HT-29 cells. A detailed understanding of the signalling network including PI3K that drives proliferation of CRC is essential for development of new anticancer drugs. Supplementary materials related to this article can be found online at doi:10.1016/j.jprot.2011.11.032.
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