Sivaneswary Chandran1, Judith Harmey PhD2, Sinead Toomey PhD2
1RCSI medical student
2Department of Molecular and Cellular Therapeutics (MCT), RCSI
Royal College of Surgeons in Ireland Student Medical Journal 2012;5: 39-45.
Introduction: Triple-negative breast cancer (TNBC) is characterised by the absence of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) on malignant cells. Insulin-like growth factors (IGFs) stimulate cell proliferation and promote cell survival in TNBC via receptor phosphorylation and activation of adaptor proteins. The aim of this project is to characterise the expression and activation of the IGF signalling pathway in a TNBC cell line, namely MDA-MB-231.
Methods: Expression of oestrogen, progesterone and growth hormone receptors and activation of the IGF signalling pathway in MDA-MB-231 cells was analysed by western blotting. The effect of stimulation with IGF1 or inhibition of epidermal growth factor receptor (EGFR)/IGF1R tyrosine kinase activity on proliferation was assessed using an MTS cell proliferation assay. Proliferation was expressed relative to untreated controls, and data was analysed by ANOVA with Tukey’s multiple comparison post hoc test.
Results: MDA-MB-231 cells express EGFR and high levels of insulin-like growth factor binding protein 4 (IGFBP4). Moreover, MDA-MB-231 cells express type I IGF1 receptors and proteins in the IGF signalling cascade, namely Erk and Akt. The presence of phosphorylated forms of these proteins suggests activation of the IGF1R signal transduction pathway in MDA-MB-231 cells. Proliferation is increased by IGF1 (E3R), a recombinant IGF1 resistant to binding by IGFBPs. Inhibition of EGFR tyrosine kinase activity or IGF1R tyrosine kinase activity inhibits proliferation of MDA-MB-231 cells.
Conclusion: These results suggest that the IGF1 signalling pathway is activated in MDA-MB-231 TNBC cells. Therefore, inhibition of the IGF1R and/or its downstream targets may be of benefit in the treatment of TNBC.
Keywords: Triple-negative breast cancer; IGF signalling pathway.
From 2005 to 2007, breast cancer was, apart from non-melanoma skin cancer, the most common cancer in women, constituting 30% of all invasive cancers.1 Breast cancer is a heterogeneous condition characterised by variable gene expression patterns. Gene expression array analysis has led to the identification of several major breast cancer subtypes, including oestrogen receptor (ER)- and progesterone receptor (PR)-positive luminal A and B, human epidermal growth receptor 2 (HER2)-positive and triple-negative breast cancer.
These subtypes have diverse histopathologic, molecular and clinical features and therefore require different therapeutic approaches.2 Triple-negative breast cancer (TNBC) is negative for ER, PR and HER2. It is typically an aggressive malignancy and has a poor prognosis. On histology, TNBC is a high-grade malignancy with a high proliferation rate and interspersed necrosis. Due to its proliferative activity, TNBC may manifest as an interval cancer, diagnosed between mammographic screenings.3 A higher incidence of TNBC is observed in African-American populations, premenopausal patients and patients with increased body weight and/or metabolic syndrome.4,5,6 Cancer cells in TNBC tend to metastasise haematogenously, and patients present with axillary lymph node metastases less often than patients with non-TNBC. Moreover, an increased rate of visceral metastasis in patients with TNBC has been documented, especially to the lungs and brain.5,7-9 Currently, TNBC is treated with conventional chemotherapy, which has limited efficacy and an unpleasant side effect profile.10-12 There is an urgent need for non-cytotoxic, targeted therapies that could prolong the lives of women with TNBC.
Limited treatment options are available for TNBC as it is unresponsive to hormonal or HER2-targeted therapies, namely tamoxifen and herceptin.10,13,14 TNBC tends to respond better than other breast cancer subtypes to neo-adjuvant chemotherapy, but patients with residual disease tend to relapse and have a poor prognosis.15 Trials with other therapeutic targets, such as EGFR, vascular endothelial growth factor, Src, MEK, PARP and histone de-acetylase inhibitors, have been initiated.3,6
Recent studies demonstrate that insulin-like growth factors (IGFs) stimulate cell proliferation and promote cell survival in TNBC by facilitating recruitment and phosphorylation of intracellular adaptor proteins.14,16-18 The signalling cascades initiated lead to the activation of proteins such as mitogen-activated protein kinase (MAPK), Akt and Erk, and ultimately to increased cell survival, proliferation and migration by modulating gene expression in the cell.15 Akt plays a key role in cell growth, proliferation, metabolism and survival.19,20 Erk is activated preferentially in response to growth factors and phorbol ester, and regulates cell proliferation and differentiation.15 Although the IGF signalling pathway has attracted considerable interest as a therapeutic target for the treatment of cancer, the effects of IGF1 inhibitors in combination with existing agents are only being tested in hormone receptor-positive or HER2-positive cancers.14,21 IGF inhibitors have not been tested on TNBC due to insufficient data from preclinical models to indicate that TNBC is IGF-responsive.15
For this reason, we aim to characterise the expression and activation of the IGF signalling pathway in the MDA-MB-231 TNBC cell line. The results herein may inform future treatment options for TNBC that target the IGF signal transduction pathway.
Materials and methods
All tissue culture was carried out using aseptic technique in a class II laminar airflow unit (LAF).22
MDA-MB-231 cells were obtained from the American Type Culture Collection (ATCC, Middlesex, UK). The base medium for this cell line is Leibovitz’s L-15 Medium (Sigma-Aldrich, MO, USA). Cells were maintained in high glucose (1g/L) Dulbecco’s Modified Eagle Medium (DMEM) (Biosera, Sussex, UK) containing 10% (v/v) FCS (Biosera) at 37°C in a humidified atmosphere of 5% CO2.
Revival of frozen cells22
Frozen cells thawed at 37°C were added to culture medium, centrifuged and the resuspended pellet incubated in fresh media overnight.
Subculture of cell lines
The cells were checked daily using an inverted microscope (Nikon Eclipse TS100, Micron Optical, Wexford, Ireland). When the cells were 70-80% confluent, they were sub-cultured.22
When the cells were 70-80% confluent, they were counted using haemocytometer, cell counter and Trypan blue dye exclusion test.22,23 The total number of viable cells was determined using the following formula:
Cells were tested monthly for mycoplasma infection using a commercial MycoAlertTM mycoplasma detection assay (Cambrex BioScience, ME, USA).
Preparation of cell lysates and protein quantification
Cells were washed in cold PBS and lysed in 100μl of a mixture of 1ml Ripa buffer (5ml Tris-HCl 1M, pH 7.4, 15ml NaCl 1M, 1ml 120% Triton x 200μl 0.5M EDTA), 10μl of 1:100 dilution protease inhibitor cocktail (Sigma) and 10μl of phosphatase inhibitor (Sigma). Lysed cells were placed on ice for 30 minutes, centrifuged at 10,000rpm for 10 minutes and stored at -80°C. Total protein was quantified using the BCA (bicinchoninic acid) assay.
Collection and concentration of conditioned medium
Culture medium was aseptically collected and stored at -80°C after the addition of protease inhibitor cocktail. Samples were concentrated using 3kDa Amicon centrifugal filters (Merck, Darmstadt, Germany).
Protein resolution was achieved by SDS-PAGE (4-20% Precise precast gel from Pierce) and transferred to nitrocellulose membranes. Membranes were blocked in TBS-T (10mM Tris-HCl, pH 7.4, 100mM NaCl, 0.1% (v/v) Tween-20) containing 5% (w/v) non-fat powdered milk for one hour and incubated overnight at 4°C with primary antibody (rabbit anti-human/mouse IGFBP4 in 5% marvel, rabbit anti-human/mouse β-Actin in 5% marvel, rabbit anti-human/mouse EGFR in 5% BSA, rabbit monoclonal anti-human/mouse pEGFR in 5% BSA, rabbit anti-human/mouse Akt in 5% BSA, rabbit anti-human/mouse pAkt in 5% BSA, rabbit anti-human/mouse IGF1R Beta in 5% BSA, and rabbit anti-human/mouse pIGF1R in 5% BSA).
Membranes were washed three times for 30 minutes with TBS-T, incubated with 1:2,000 horseradish peroxidase-conjugated anti-rabbit antibody (DAKO, Glostrup, Denmark) in TBS-T/5% (w/v) non-fat powdered milk for one hour, then washed three times for 30 minutes. Specific bands were illustrated by ECL re-agent treatment followed by exposure to x-ray film.
PAPP-A RT(Reverse transcriptase)-PCR
PAPP-A RT-PCR was conducted followed by agarose gel electrophoresis of RNA.24
MDA-MB-231 cells express EGFR and low levels of HER3, but are negative for ER, PR and HER2 (). MDA-MB-231 cells express high levels of IGFBP4 (Figure 1a), and low levels of PAPP-A (Figure 2). Moreover, they express IGF1R (Figure 1a) and proteins along the IGF signalling cascade; namely, Erk and Akt (Figure 1b). Proliferation was increased by recombinant IGF1 (E3R), but not by wild-type IGF1 (Figure 3).
Inhibition of EGFR tyrosine kinase activity or IGF1R tyrosine kinase activity by lapatinib inhibited proliferation (Figure 4a), and a similar effect was observed with dual inhibition of PI3K/mTOR using BEZ235 or Akt/P70S6K using AT7867 (Figures 4d and 4e). Higher concentration of IGF1R inhibitors (AG1024 and PPP) inhibited cell growth (Figures 4b and 4c). Herceptin and MAPK inhibitor (PD98050) have no effect on proliferation in MDA-MB-231 cells.
The IGF signalling pathway is highly implicated in breast cancer, and IGF1R is overexpressed in most breast malignancies.25,26 Expression of IGF1R by MDA-MB-231 cells suggests that the IGF signal transduction pathway may play an important role in controlling cell proliferation and cell survival in TNBC. The presence of phosphorylated IGF1R and downstream proteins in the IGF cascade suggests that the IGF signalling pathway is activated in MDA-MB-231 cells.
In this investigation, IGF1 stimulation did not increase proliferation in MDA-MB-231 cells. This may be due to expression of high levels of IGFBP4 and low levels of PAPP-A in the cells. IGFBP4 binds to IGF1, thus making it unavailable to stimulate proliferation. The protease PAPP-A cleaves to IGFBP4 releasing biologically active IGF1. However, stimulation with recombinant IGF1 (E3R) increased cell proliferation. Recombinant IGF1R (E3R) is resistant to binding by IGFBPs and is free to promote cell proliferation.25
Treatment with lapatinib (tyrosine kinase inhibitor) inhibited cell proliferation. Lapatinib inhibits receptor signal processes by binding to the ATP-binding pocket of the EGFR protein kinase domain, thus preventing self-phosphorylation and subsequent activation of the signalling pathway.27 Dual inhibitors of PI3K/mTOR (BEZ235) or Akt/P70S6K (AT7867) blocked proliferation in MDA-MB-231 cells. mTOR is a cell cycle regulator and a downstream effector in the PI3K/PTEN/Akt pathway. Loss of phosphatase and tensin homolog (PTEN) occurs frequently in TNBC, which leads to increased Akt and mTOR activation.28
Thus, it is possible that the inhibition of mTOR may be a therapeutic target in TNBC management. The results demonstrate that the IGF1 signalling pathway is activated in MDA-MB-231 TNBC cells. Receptors and downstream signal transduction proteins are present and phosphorylated in response to IGF1. Therefore, inhibition of the IGF1R and/or its downstream targets may be of benefit in the treatment of TNBC.
To gain a better understanding of the activation of the IGF signalling pathway and the effects of inhibiting the pathway, it may be of benefit to use a panel of TNBC cell lines in future studies. Since this study focuses on a human cell line, the results may not be applicable in animal model studies. The expression of EGFR, IGF1R and proteins along the IGF signalling pathway should be investigated in response to treatment with IC50 doses of inhibitors. It will also be of benefit to investigate the effects of co-inhibition of IGF1R and other proteins along IGF signalling cascade. The role of IGF1R in non-mitogenic responses in TNBC, such as cell migration and invasion, should also be determined.
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