The authors thank the UK Medical Research Council and pharmaceutical companies that support the Division of Signal Transduction Therapy (DSTT) at University of Dundee (AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Merck-Serono and Pfizer) for financial support. The Oncomine data (Supporting Table S1) was searched as part of a collaboration with AstraZeneca. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. AstraZeneca had no further role in the study design, data collection and analysis, nor in the decision to publish, nor preparation of the manuscript.
Conceived and designed the experiments: GH KD CM. Performed the experiments: GH KD CM. Analyzed the data: GH KD CM. Contributed reagents/materials/analysis tools: GH KD CM. Wrote the paper: GH CM.
Insulin receptor substrate 1 (IRS1) and IRS2 are well-characterized adapter proteins that relay signals from receptor tyrosine kinases to downstream components of signalling pathways. In contrast, the function of IRS4 is not well understood. IRS4 overexpression has been associated with acute lymphoblastic leukaemia and subungual exostosis, while point mutations of IRS4 have been found in melanomas. Here, we show that while IRS4 expression is low in most cancer cell lines, IRS4 mRNA and protein levels are markedly elevated in certain cells including the NCI-H720, DMS114, HEK293T and HEK293AAV lines. Surprisingly, IRS4 expression was also strongly induced when HEK293 cells were infected with retroviral particles and selected under puromycin, making IRS4 expression a potential off-target effect of retroviral expression vectors. Cells with high expression of IRS4 displayed high phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels, as well as elevated Akt and p70 S6 kinase activities, even in the absence of growth factors. PI 3-kinase (PI3K) signalling in these cells depends on IRS4, even though these cells also express IRS1/2. Knockdown of IRS4 also inhibited cell proliferation in cells with high levels of IRS4. Together, these findings suggest IRS4 as a potential therapeutic target for cancers with high expression of this protein.
The insulin receptor substrate (IRS) proteins are a family of cytoplasmic adaptors that couple activation of the insulin receptor and other receptor tyrosine kinases to downstream PI3K–Akt and Ras signalling pathways
In humans, point mutations of IRS4 and overexpressions of IRS4 due to chromosomal translocations, were recently identified in human paediatric T-cell acute lymphoblastic leukaemia and subungual exostosis, a benign tumour of bone and cartilage in the distal phalanges of fingers and toes
In this study we show that while expression of IRS4 is generally low in the studied panel of cancer cell lines, it is high in NCI-H720, DMS114, HEK293T and HEK293AAV cells and that PI3K signalling in these cell lines relies on IRS4, but not IRS1. We also found that IRS4 expression is strongly induced upon infection of HEK293 cells with retroviral particles and subsequent selection with puromycin. Our findings also indicate that high expression of IRS4 has a significant role in PI3K signalling and therefore could be exploited to target this pathway in certain types of cancer.
In comparison to other members of the IRS family, IRS4 is not as widely expressed
(A) The graph represents the IRS4 mRNA expression in 298 cancer cell lines. Data in Oncomine (
To establish whether the high IRS4 expression in the aforementioned four cell lines is functional, we performed siRNA-mediated knockdown of IRS4 or IRS1 (
(A) Cell lines exhibiting high levels of IRS4 such as DMS114, NCI-H720, HEK293AAV, HEK293T were transfected with 100 nM final concentration of control (Scrambled), IRS4 or IRS1 siRNAs using Dharmafect 1 transfection reagent following manufacturer's instructions. After 48 h, cells were harvested and lysates (20 µg) were immunoblotted using the indicated antibodies to test the activation status of the PI3K pathway. (B) As in (A) except that the experiment was performed in cells with low expression of IRS4 such as HeLa, A431, HEK293, MDA-MB-231 and A549.
(A) DMS114, (B) HEK293T and (C) 293AAV cells were transfected with control (Scrambled) and IRS4 siRNA oligos. At 24 h post transfection, cells were seeded in 24-well plates (20 000 cells per well). For each time point, cells were washed with phosphate-buffered saline (PBS), fixed in 4% (v/v) paraformaldehyde in PBS for 15 min, washed in water once, stained with 0.1% Crystal Violet in 10% ethanol for 20 min and washed thrice with water. Crystal Violet was extracted from cells with 0.5 ml of 10% (v/v) acetic acid for 20 min while shaking at room temperature and the absorbance measured at 590 nm.
HEK293 cells have very low IRS4 protein levels (
(A) Clones of HEK293 cells expressing different levels of IRS4 (#1-low, #2- and #3-high) obtained after retroviral infection and puromycin selection as described in materials and methods, were serum starved and then stimulated with IGF1 or 10% FBS. Cell lysates (30 µg) were subject to immunoblotting with the indicated antibodies. (B) As in (A), except that cells were starved of amino acids and then stimulated with amino acids or 10% FBS for 20 min. (C and D) The cells expressing different levels of IRS4 were serum deprived overnight and assayed for the catalytic activity of immunoprecipitated endogenous Akt and S6K1 using Crosstide peptide as substrate, as described in materials and methods. (E) Clones #1 and #3 of HEK293 cells were serum starved overnight and assayed for PIP3 as in
To test whether the increases in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels, and Akt and S6K activities, were due to IRS4, we knocked down IRS4 or IRS1 using siRNA.
U2OS-FlpIn™ cells stably expressing FLAG-tagged IRS4 also displayed strongly enhanced PI3K signalling, compared to control empty vector cells (
(A) U2OS-Flpin™ cells expressing empty vector or IRS4-FLAG protein were analysed for the activation of PI3K pathway components under serum starved (SS), amino acid-starved (-AA) and 10% FBS conditions. Cells were deprived of serum for 12 h (SS) and starved of amino in EBSS for 2 h (-AA). (B) U2OS-Flpin™ cells expressing empty vector, full length IRS4-FLAG (WT), and the indicated fragments containing residues from 200–end-IRS4-FLAG or 336–end-IRS4-FLAG proteins were analysed for the activation of PI3K pathway components, in the presence or absence of serum. (C) As in B, except that the cells were treated with IGF1 (50 ng/ml, 20 min). The FLAG tagged proteins expressed were analysed for the Tyrosine phosphorylation as well as the binding to the p85 regulatory subunit of PI3 kinase.
Interestingly, IRS4 mutants lacking PH, or PH and PTB domains showed some tyrosine phosphorylation and also interacted with the p85 subunit of PI3K, and these interactions were enhanced by IGF1, although no increase in tyrosine phosphorylation was observed in response to IGF1 or 10% serum. The tyrosine phosphorylation of the mutant lacking PH domain was more difficult to assess since the signal interfered with an unspecific band recognized by the phosphotyrosine antibody. Strikingly, full length IRS4 or IRS4 lacking both PH and PTB domain were localized to the cytoplasm and plasma membranes under both serum starved and IGF1-stimulated conditions, whereas IRS4 lacking only the PH domain was localized to the nucleus under serum starved conditions (
U2OS-Flpin™ cells expressing full length IRS4-FLAG and the indicated fragments containing residues from 200–end-IRS4-FLAG or 336–end-IRS4-FLAG proteins were analysed for their subcellular localization, under serum starved (SS) or addition of IGF1(50 ng/ml, 20 min). Cells were stained for FLAG antibody and with DAPI as described in Materials and Methods.
Considering that the relative roles of IRS4 may differ in the rodents used for genetic studies, and humans, which lack IRS3, we wished to define how this protein family evolved. The vertebrate IRS proteins displays the genetic signature of a family of 2R-ohnologues, which are protein families generated by the two rounds of whole-genome duplication (2R-WGD) at the origin of the vertebrates
A circle plot links the human gene synteny clusters containing human IRS1, IRS2, IRS4 genes and the IRS3P pseudogene. Plots were generated using the Synteny Database (teleost.cs.uoregon.edu/synteny_db)
A recent evolutionary analysis placed IRS5/Dok4 (downstream of kinase-4) and IRS6/Dok5 in the same family as IRS1, IRS2, IRS3 and IRS4
Our results caution that IRS4 expression is a potential off-target effect of retroviral delivery of genetic material into cells, and that high IRS4 levels identified in certain cancer cells can drive signalling via PI3K even in the absence of growth factors. Our findings suggest that cells with high levels of IRS4 use this protein for activation of PI3K signalling, even though they also contain IRS1 and IRS2. Perhaps, cells expressing IRS4 build an accompanying regulatory network that cannot accommodate IRS1 and/or IRS2, so that this network collapses when the IRS4 is subsequently removed. An alternative suggestion is that the expression of IRS4 triggers a feedback mechanism that inhibits the action of IRS1 and IRS2
We found that when IRS4 is overexpressed in cells, the PI3K pathway is activated to the extent seen upon stimulation of cells with IGF1 (50 ng/ml for 20 min) (
Lack of the PH domain (up to residue 200) caused IRS4 to be localized predominantly in the nucleus (
Previously, IRS4 levels were shown to be elevated after adenovirus 5 E1A infection and transfections of cells
Protease inhibitor cocktail tablets (#1697498) were from Roche; PEI from Polysciences; Dharmafect 1 from Thermo Scientific; Protein G-Sepharose and enhanced chemiluminescence Western blotting kit were from Amersham Bioscience; insulin, transferrin, sodium selenite, hydrocortisone, beta-oestradiol and amino-6-methylmercaptopurine were from Sigma. Precast NuPAGE polyacrylamide Bis-Tris gels, Colloidal Coomassie, LDS sample buffer, hygromycin, tetracycline, zeocin, GIBCO® Earle's Balanced Salt Solution (EBSS) and Gibco® MEM Amino Acids Solution 50 were from Invitrogen; sequencing-grade trypsin was from Promega; and microcystin-LR was kindly provided by Dr. Linda Lawton (Robert Gordon University, Aberdeen, UK).
The IRS4 antibody was from Santa Cruz Biotechnology (sc-28830) and IRS2 antibody from Millipore (MABS15). Antibodies that recognize pSer473 Akt, pThr308 Akt, pThr389-S6K, pSer235/6-S6, pThr1135-rictor, phosphotyrosine, GAPDH and IRS1 were from Cell Signaling Technology. Anti-FLAG M2 was from Sigma. Anti-Akt was raised in sheep (S695B) against the RPHFPQFSYSASGTA peptide (residues 466–480 of human Akt); and anti-S6K was raised in sheep (S417B) against the peptide AGVFDIDLDQPEDAGSEDEL (residues 25–44 of human S6K). Antibody production and purification were arranged via the Division of Signal Transduction Therapy (DSTT) of the University of Dundee.
Cells were starved of amino in EBSS for 2 h (-AA). A mixture of amino acids (50X Gibco® MEM Amino Acids Solution) was used at 1× concentration for 10 min.
Cells were lysed in 0.35 ml per 10 cm dish in ice-cold Triton X-100 Lysis Buffer comprising 25 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM benzamidine, 0.2 mM PMSF, 0.1% 2-mercaptoethanol, 1 μM microcystin-LR, 0.27 M sucrose and one mini Complete™ protease inhibitor cocktail tablet (#1697498, Roche) per 10 ml of lysis buffer. Lysates were clarified by centrifugation (16,000× g at 4°C for 15 min); snap frozen and stored at −80°C. Protein concentrations were determined with Coomassie Protein Assay Reagent (Thermo Scientific).
Cells were transfected with control (Scrambled) and IRS4 siRNA oligos. At 24 h post-transfection, cells were seeded in 24-well plates (20000 cells per well). For each time point, cells were washed with PBS, fixed in 4% (v/v) paraformaldehyde in PBS for 15 min, washed with water once, stained with 0.1% Crystal Violet in 10% ethanol for 20 min and washed thrice with water. Crystal Violet was extracted from cells with 0.5 ml of 10% (v/v) acetic acid for 20 min while shaking at room temperature and the absorbance measured at 590 nm.
Recombinant DNA procedures, restriction digests, ligations and PCR were performed using standard protocols by Dr Rachel Toth in the DSTT. All PCR reactions were carried out using KOD Hot Start DNA polymerase (Novagen). DNA sequencing was performed by The Sequencing Service, College of Life Sciences, University of Dundee (
U2OS-Flpin™ cells maintained in DMEM containing 10% FBS and 2 mM L-glutamine, were transfected with pcDNA5-FRT-TO vectors to express IRS4-FLAG or IRS4-FLAG fragments together with the plasmid pOG44 (Invitrogen), which encodes Flp recombinase. The pOG44 to pcDNA5-FRT-TO vector ratio was (9∶1). At 48 h post transfection, the cells were selected in DMEM growth medium containing 100 μg/ml hygromycin B.
Kinase assays were performed as in
Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) measurements were kindly performed by Dr. Alexander Gray (University of Dundee) as previously described
Cells grown on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 for 5 min, rinsed with PBS, blocked in 10% donkey serum in PBS for 30 min and incubated with primary antibody (FLAG-M2 (1 µg/ml)) in 1% BSA/PBS overnight at 4°C. The coverslips were washed 3 times with 1% BSA/PBS and incubated with secondary antibodies (Alexa Fluor® 594 Donkey Anti-Mouse IgG (1∶500)) for a further hour at room temperature in the dark. After 3 washes with 1% BSA in PBS, cells were stained with DAPI (4′, 6-diamidino-2-phenylindole) and mounted using Vectashield (Vector Laboratories). Cells were visualized with a Nikon Eclipse Ti-S fluorescence microscope at 40× magnification. Images were captured by NIS-Elements software and processed using Adobe Photoshop.
All cells were maintained at 37°C in a humidified atmosphere with 5% CO2. NCI-H720, HuNS1, DMS114 and ES-2 cells were obtained from ATCC. NCI-H720 cells were maintained in DMEM: F12 medium containing 5 µg/ml insulin, 10 µg/ml transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM beta-oestradiol, 2 mM L-glutamine and 5% FBS; HuNS1 cells were in RPMI 1640 medium containing 2 mM L-glutamine, 4.5 g/l glucose, 10 mM HEPES, 1 mM sodium pyruvate, 1.5 g/l sodium bicarbonate, 20 µM 2-amino-6-methylmercaptopurine and 15% FBS; DMS114 were maintained in Waymouth's MB medium containing 10% FBS; ES-2 cells in minimum essential medium Eagle alpha modification (αMEM) supplemented with 5% FBS; and HEK293, HEK293AAV, HEK293T, HeLa and U2OS cells were grown in DMEM containing 10% FBS supplemented with 2 mM L-glutamine and 1% penicillin/streptomycin.
H29, a packaging cell line for retrovirus production, was maintained in DMEM containing 10% FBS, 2 μg/ml puromycin, 0.3 mg/ml G418 and 0.5 µg/ml tetracycline. Before transfection, the cell medium was changed to DMEM containing 10% FBS only. Cells grown in T75 flasks to 50% confluency were transfected with 15 µg of shRNA retroviral vector (pSuper-retro.puro) and 45 μl PEI in 2 ml Opti-MEM. After 48 h, viruses were collected, filtered (0.45 μm pore size) and used to infect HEK293 cells in the presence of 5 μg/ml polybrene. At 24 h post infection, the culture medium was replaced and cells were selected in DMEM containing 10% FBS and 3 µg/ml puromycin.
siRNA(ON-TARGETplus SMARTpool) oligos towards IRS4 and IRS1 and Scrambled (control) were from Thermo Scientific. Cells at 40–50% confluency were transfected using Dharmafect 1 transfection reagent following manufacturer's instructions. Briefly, for transfection of cells in one well of a 6-well plate, siRNA oligos (100 nM final concentration) and Dharmafect 1 (10 µl) were incubated separately in 200 µl of Opti-MEM for 5 min, mixed gently and incubated at room temperature for a further 20 min. The mixture was added slowly to the cells and the culture media replaced with fresh after 12 h of siRNA transfection.
Results shown are representative of at least three similar experiments with the exception of
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We are grateful to Dr Alexander Gray for measuring PIP3. We thank Drs John Mclauchlan and Ana Filipe, University of Glasgow, for lysates of cells transfected with hepatitis C; the antibody production teams managed by Drs James Hastie and Hilary McLauchlan of the Division of Signal Transduction Therapy (DSTT), University of Dundee; Dr Rachel Toth of the DSTT for cDNA constructs, the DNA Sequencing Service at the College of Life Sciences, University of Dundee; and Kirsten Mcleod and Janis Stark for tissue culture support.