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Sivaneswary Chandran1, Judith Harmey PhD2, Sinead Toomey PhD2

Author affiliations
1RCSI medical student
2Department of Molecular and Cellular Therapeutics (MCT), RCSI

Royal College of Surgeons in Ireland Student Medical Journal 2012;5: 39-45.

Abstract
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.


Introduction

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

Cell culture
All tissue culture was carried out using aseptic technique in a class II laminar airflow unit (LAF).22

Cell lines
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

Cell counting
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:

Mycoplasma testing
Cells were tested monthly for mycoplasma infection using a commercial MycoAlertTM mycoplasma detection assay (Cambrex BioScience, ME, USA).

Protein analysis
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).

Western blotting
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

Results

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).

FIGURE 1: Western blot analysis in MDA-MB-231 cells. FIGURE 1a: Expression of oestrogen, progesterone and growth hormone receptors in MDA-MB-231 cells. (a) Oestrogen receptor (ERα) expression in MDA-MB-231 cell lysate. Oestrogen-responsive MCF-7 cells served as positive control. (b) Progesterone receptor (PR) expression in MDA-MB-231 cells. MCF-7 cell lysate served as positive control. (c) HER2 expression in MDA-MB-231 cells. SK-BR-3 cells served as positive control. (d) HER3 expression in MDA-MB-231 cells. SK-BR-3 cell lysate served as positive control. (e) EGFR expression in MDA-MB-231 cells. SK-BR-3 cells served as positive control. (f) IGF1R expression in MDA-MB-231 cells. 4T1.2 cells served as positive control. (g) IGFBP4 expression in cell lysate (Lys) and conditioned medium (CM) from MDA-MB-231 cells. Recombinant human IGFBP4 served as positive control. (h) β-Actin expression as control for sample loading.


FIGURE 1(b): Expression of proteins along IGF signalling pathway in MDA-MB-231 cells. (a) Akt expression in MDA-MB-231 cells. 4T1.2 cell lysate served as positive control. (b) Erk expression in MDA-MB-231 cells. SK-BR-3 cells served as positive control. (c) P-Akt expression in MDA-MB-231 cells. 4T1.2 cell lysate served as positive control. (d) P-Erk expression in MDA-MB-231 cells. SK-BR-3 cells served as positive control. (e) P-IGF1R expression in MDA-MB-231 cells. 4T1.2 cells served as positive control.


FIGURE 2: Reverse transcriptase-PCR (RT-PCR) of PAPP-A. PAPP-A was amplified by RT-PCR from cDNA prepared from HEK293 cells (positive control) and MDA-MB-231 cells. MDA-MB-231 cells amplified without reverse transcriptase were used as a negative control. β-Actin was amplified from all samples to control for equal loading and sample quality.


FIGURE 3: Effect of IGF1 on MDA-MB-231 cells. (a) IGF1 stimulation on MDA-MB-231 cells. Cells were treated in triplicate with IGF1 and proliferation was assayed by MTS assay. (b) IGF1 (E3R), a recombinant IGF1 stimulation on MDA-MB-231 cells. Cells were treated in triplicate with IGF1 (E3R) and proliferation was assayed by MTS assay. Proliferation is expressed as a percentage of control where control is 100%. Data (n=2) are expressed as mean ± S.E.M.* and analysed by ANOVA with Tukey’s multiple comparison post hoc test. *S.E.M. = Standard error of the mean.

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.

FIGURE 4: Effect of inhibitors on MDA-MB-231 cells. FIGURE 4a: Effect of lapatinib (tyrosine kinase inhibitor) on MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with increasing (0-10μM) concentrations of lapatinib for 72 hours, before proliferation was measured by MTS assay. Proliferation is expressed as percentage of control where control is 100%. Data are expressed as mean ± S.E.M. and analysed by ANOVA with Tukey’s multiple comparison post hoc test. *P<0.05 vs. 0μM; $P<0.05 vs. 0.1μM. (b) Effect of lapatinib on MDA-MB-231 cell proliferation expressed as log10. IC50 = 3.5μM.


FIGURE 4b: Effect of AG1024 (IGF1R inhibitor) on MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with increasing (0-10μM) concentrations of AG1024 for 72 hours, before proliferation was measured by MTS assay. Proliferation is expressed as percentage of control where control is 100%. Data are expressed as mean ± S.E.M. and analysed by ANOVA with Tukey’s multiple comparison post hoc test. (b) Effect of AG1024 on MDA-MB-231 cell proliferation expressed as log10. IC50 = 6μM.


FIGURE 4c: Effect of PPP (IGF1R inhibitor) on MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with increasing (0-10μM) concentrations of PPP for 72 hours, before proliferation was measured by MTS assay. Proliferation is expressed as percentage of control where control is 100%. Data are expressed as mean ± S.E.M. and analysed by ANOVA with Tukey’s multiple comparison post hoc test. (b) Effect of PPP on MDA-MB-231 cell proliferation expressed as log10. IC50 = 5μM.


FIGURE 4d: Effect of AT7867 (Akt/P70S6K inhibitor) on MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with increasing (0-10μM) concentrations of AT7867 for 72 hours, before proliferation was measured by MTS assay. Proliferation is expressed as percentage of control where control is 100%. Data are expressed as mean ± S.E.M. and analysed by ANOVA with Tukey’s multiple comparison post hoc test. (b) Effect of AT7867 on MDA-MB-231 cell proliferation expressed as log10. IC50 = 5μM.


FIGURE 4e: Effect of BEZ235 (PI3K/mTOR inhibitor) on MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with increasing (0-10μM) concentrations of BEZ235 for 72 hours, before proliferation was measured by MTS assay. Proliferation is expressed as percentage of control where control is 100%. Data are expressed as mean ± S.E.M. and analysed by ANOVA with Tukey’s multiple comparison post hoc test. *P<0.05 vs. 0μM; **P<0.01 vs. 0μM; ***P<0.001 vs 0μM. (b) Effect of BEZ235 on MDA-MB-231 cell proliferation expressed as log10. IC50 = 4.2μM.

Discussion

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.

References

  1. National Cancer Registry [homepage on the Internet]. Cancer in Ireland 1994-2007 – Summary. Annual Statistical Report of the National Cancer Registry 2009 – 8 pages. Available from: http://www.ncri.ie/pubs/pubfiles/summary_report_19942007.pdf.
  2. Sørlie T, Perou CM, Tibshirani R et al. Gene expression patterns of breast carcinomas distinguish tumour subclasses with clinical implications. Proc Natl Acad Sci USA. 2001;98(19):10869-74.
  3. De Laurentiis M, Cianniello D, Caputo R, Stanzione B, Arpino G, Cinieri S et al. Treatment of triple negative breast cancer (TNBC): current options and future perspectives. Cancer Treat Rev. 2010;36(S3):S80-S86.
  4. Pal SK, Mortimer J. Triple-negative breast cancer: novel therapies and new directions. Maturitas. 2009;63:269-74.
  5. Aksoy S, Dizdar O, Harputluoglu H, Altundag K. Demographic, clinical, and pathological characteristics of Turkish triple-negative breast cancer patients: single center experience. Ann Oncol. 2007;18:1904-6.
  6. Gluz O, Liedtke C, Gottschalk N, Pusztai L, Nitz U, Harbeck N. Triple-negative breast cancer – current status and future directions. Ann Oncol. 2009;20:1913-27.
  7. Arslan C, Dizdar O, Altundag K. Pharmacotherapy of triple-negative breast cancer. Expert Opin Pharmacorther. 2009;10(13):2081-93.
  8. Dent R, Trudeau M, Pritchard KI et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007;13(15):4429.
  9. Van Calster B, Vanden Bempt I, Drijkoningen M et al. Axillary lymph node status of operable breast cancers by combined steroid receptor and HER-2 status: triple positive tumours are more likely lymph node positive. Breast Cancer Res Treat. 2009;113:181-7.
  10. Schneider BP, Winer EP, Foulkes WD, Garber J, Perou CM, Richardson A et al. Triple-negative breast cancer: risk factors to potential targets. Clin Cancer Res. 2008;14(24):8010-8.
  11. Banerjee S, Reis-Filho JS, Ashley S, Steele D, Ashworth A, Lakhani SR et al. Basal-like breast carcinomas: clinical outcome and response to chemotherapy. J Clin Pathol. 2006;59:729-35.
  12. Sirohi B, Arnedos M, Popat S, Ashley S, Nerurkar A, Walsh G et al. Platinum-based chemotherapy in triple-negative breast cancer. Ann Oncol. 2008;19(11):1847-52.
  13. Klinakis A, Szabolcs M, Chen G, Xuan S, Hibshoosh H, Efstratiadis A. IGF1r as a therapeutic target in a mouse model of basal-like breast cancer. Proc Natl Acad Sci USA. 2008;105(49):19378-83.
  14. Pollak M. Insulin and insulin-like growth factor signalling in neoplasia. Nat Rev Cancer. 2008;8(12):915-28.
  15. Davison Z, de Blacquire GE, Westley BR, May FEB. Insulin-like growth factor-dependent proliferation and survival of triple-negative breast cancer cells: implications for therapy. Neoplasia. 2011;13(6): 504-15.
  16. Hankinson SE, Willett WC, Colditz GA, Hunter DJ, Michaud DS, Deroo B et al. Circulating concentrations of insulin-like growth factor-1 and risk of breast cancer. Lancet. 1998;351(9113):1393-6.
  17. Schernhammer ES, Holy JM, Pollak MN, Hankinson SE. Circulating levels of insulin-like growth factors, their binding proteins, and breast cancer risk. Cancer Epidemiol Biomarkers Prev. 2005;14(3):699-704.
  18. Renehan AG, Harvie M, Howell A. Insulin-like growth factor (IGF)-1, IGF binding protein-3, and breast cancer risk: eight years on. Endocr Relat Cancer. 2006;3(2):273-8.
  19. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer. 2002;2:489-501.
  20. Yap TA, Garrett MD, Walton MI, Raynaud F, de Bono JS, Workman P. Targeting the PI3K-AKT-mTOR pathway: progress, pitfalls, and promises. Curr Opin Pharmacol. 2008;8:449-557.
  21. Gualberto A. Figitumumab (CP-751, 871) for cancer therapy. Expert Opin Biol Ther. 2010;10(4):575-85.
  22. Life Technologies. GIBCO® Cell Culture Basics. [homepage on the Internet]. Cited December 2, 2011. Available from: http://www.invitrogen.com/site/us/en/home/References/gibco-cell-c ulture-basics.html.
  23. Strober W. Trypan Blue exclusion test of cell viability. Current Protocols in Immunology. 2001;A.3B.1-A.3B.2.
  24. Ryan AJ, Napoletano S, Fitzpatrick PA, Currid CA, O’Sullivan NC, Harmey JH. Expression of a protease-resistant insulin-like growth factor-binding protein-4 inhibits tumour growth in a murine model of breast cancer. Br J Cancer. 2009;101(2):278-86.
  25. Huynh HT, Tetenes E, Wallace L, Pollak M. In vivo inhibition of insulin-like growth factor I gene expression by tamoxifen. Cancer Res. 1993;53:1727-30.
  26. Peyrat JP, Bonneterre J, Hecquet B, Vennin P, Louchez MM, Fournier C et al. Plasma insulin-like growth factor-1 (IGF-1) concentrations in human breast cancer. Eur J Cancer. 1993;29A:492-7.
  27. Nelson MH, Dolder CR. Lapatinib: a novel dual tyrosine kinase inhibitor with activity in solid tumors. Ann Pharmacother. 2007;40(2):261-9.
  28. Bosch A, Eroles P, Zaragoza R, Viña JR, Lluch A. Triple-negative breast cancer: molecular features, pathogenesis, treatment and current lines of research. Cancer Treatment Reviews. 2010;36:206-15.

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