Background
Worldwide, more than 1.7 million women are diagnosed annually with breast cancer of whom at least 500,000 die as a result of metastatic disease [
1]. Metastasis necessitates release of malignant cells from the primary tumour and their movement to and establishment at distant sites. Normal breast epithelial and myoepithelial cells attach to each other and to the extracellular matrix. Loss of these attachments induces programmed cell death in a process called anoikis [
2‐
4]. Breast cancer cells must become resistant to anoikis as they invade breast and surrounding tissue, intravasate into blood and lymphatic vessels and metastasise [
5]. Blockade of the pathways responsible for anoikis resistance offers a powerful strategy for the elimination of metastatic cells.
Integrins are transmembrane proteins that provide connections between extracellular matrix proteins and the actin-based cytoskeleton in normal tissues. Integrins transduce signals via sub-membrane, focal adhesion protein complexes, called focal adhesions, which connect integrins with signal transduction proteins [
6,
7]. Integrin engagement activates focal adhesion kinase (FAK) to suppress anoikis [
3] by transmission of cell survival signals through multiple signal transduction pathways [
3,
8].
The effects of insulin-like growth factors (IGFs) are mediated by the transmembrane type I IGF or insulin receptors and multiple intracellular signal transduction pathways [
9]. Work with transgenic animals implicates IGFs in carcinogenesis [
10] and they are significant regulators of breast cancer cell proliferation and invasion [
11‐
15]. Consequently, the IGF signal transduction pathway has been identified as a therapeutic target and inhibitors of the type I IGF receptor have been developed by pharmaceutical companies [
9,
16‐
21].
IGFs confer anoikis resistance in embryonic fibroblasts that have been engineered to overexpress the type I IGF receptor [
22] but the importance of IGFs in anoikis resistance and the mechanism by which they might act in oestrogen-responsive breast cancer is unknown. Similarly, the signal transduction pathways involved in the induction of anoikis and in anoikis resistance of breast cancer cells have not been determined.
There have been a few studies that purport to examine the effects of the IGF signal transduction pathway upon anoikis breast cancer cells. However, caspase-dependent programmed cell death was not measured in these studies. In immortalized, normal MCF710A cells that have been modified to overexpress the type I IGF receptor, PTK6 increased signal transduction through the type I IGF receptor and IRS-1 increased the number of viable cells grown in unattached conditions [
23]. Another study reported that effects of IGF on the ratio of isoforms of the C/EBPβ protein reduced the proportion of unattached MCF710A cells in sub-G1 phase [
24]. Disruption of the type I IGF receptor signal transduction pathway decreased numbers of viable cells of a metastatic variant of MDA-MB-435 breast cancer cell line grown as unattached cells [
14]. Another study reported that activation of the p53 pathway after MCF-7 cell detachment leads to a caspase-independent reduction in mitochondrial activity, and that calveolin may reverse the reduction
via an increase in type I IGF receptor [
25]. Thus, despite the impression conveyed in the titles or abstracts of these articles, there have been no studies of the effects of IGFs on anoikis in human breast cancer cells.
We have shown that IGFs inhibit apoptosis in triple-negative breast cancer cells [
12] which suggested that they could protect against breast cancer cell anoikis and that blockade of the IGF signal transduction pathway might offer a strategy for promoting anoikis and reducing metastasis. The overall aim of the current study was to investigate the mechanisms by which oestrogen-responsive breast cancer cells evade anoikis. We established an
in vitro model of anchorage-independent, caspase-dependent cell death and investigated the changes in intracellular signal transduction involved, whether IGF-1 protects the cells from anoikis and the receptor and signal transduction pathway through which IGFs act.
Discussion
The majority of breast cancer deaths are caused by the effects of distant metastases in vital organs. Metastatic malignant cells accrue a plethora of characteristics in distinct phases: metastasis initiation, progression and virulence [
5]. They must be able to survive without normal cellular and cell matrix attachments to initiate metastasis. The development of resistance to anoikis, the apoptotic process triggered by inappropriate or absence of cell adhesion is an important facet of this transition.
Improvements in the management and survival of breast cancer patients and identification of therapeutic targets will be facilitated by a more profound understanding of metastasis. To this end, we have established a model of oestrogen-responsive breast cancer cell anoikis. Anoikis can be mediated through extrinsic, intrinsic or caspase-independent apoptotic pathways [
37,
38]. There have been limited studies on anoikis in breast cancer. Breast cancer cells as exemplified by Hs578T, MDA-MB-231, BT-474 and T-47D have been reported to be inherently resistant to anoikis and it was concluded that this resistance does not involve the PI3-kinase/Akt or Grb2/Ras/MAP-kinase pathways [
39]. In contrast, our experiments have identified conditions that allow analysis of breast cancer cell anoikis resistance and have highlighted the importance of the intrinsic apoptotic pathway.
Several signal transduction pathways implicated in anoikis emanate from the focal adhesion complex in response to integrin-mediated cell adhesion to the extracellular matrix. Amongst these, phosphorylated FAK interacts with Grb2 and PI3-kinase [
40‐
42] to activate the Ras/MAP-kinase and Akt pathways, respectively [
41,
43]. Our data emphasise the importance of the PI3-kinase/Akt pathway in oestrogen-responsive breast cancer as the rapid and dramatic loss of FAK phosphorylation within 15 minutes of detachment, followed by PARP cleavage after 1 hour, was associated with a decrease in phosphorylated Akt but an increase in phosphorylated MAP-kinases. Consistent with involvement of the intrinsic pathway, Bad phosphorylation decreased concomitantly with phosphorylated Akt and caspase 3 was activated. Dephosphorylated Bad forms heterodimers with Bcl-2 and Bcl-X
L, thereby preventing them from interacting with and inhibiting Bax and Bak, which would otherwise form pores in the mitochondrial membrane to release cytochrome c and trigger cell death [
38].
Our data show that breast cancer cell culture medium contains factors that confer anoikis resistance and that the major factor is a ligand that signals through the type I IGF receptor. A protective effect of IGF-1 against anoikis was demonstrated first in mouse embryonic fibroblasts engineered to overexpress the type I IGF receptor [
22]. As far as we are aware, this is the first demonstration that IGFs protect breast cancer cells from caspase-dependent anoikis.
Many mechanisms are suggested to explain how malignant cells that detach from primary tumours evade cell death. These include changes to integrin expression [
44], hypoxia which induces ligand-independent activation of growth factor receptors and redox-mediated decrease of pro-apoptotic factors [
45], and EMT activation [
46]. Our results demonstrate that oestrogen-responsive breast cancer cells avoid anoikis
via a prosurvival pathway in which the type I IGF receptor is activated by IGFs. The type I IGF receptor is expressed widely on breast cancer cells and mediates the effects of IGFs on cell migration [
13,
47] and proliferation [
11,
12,
48]. IGF-1 activated both the PI3-kinase/Akt and Grb2/Ras/MAP-kinase pathways but our experiments with PI3-kinase, Akt and MEK inhibitors establish that anoikis resistance is conferred preferentially through the PI3-kinase/Akt pathway.
Although the Grb2/Ras/MAP-kinase pathway is not involved in the anoikic resistance of oestrogen-responsive breast cancer cells, its inhibition induces significant cell death which is abrogated almost completely by IGF-1. IGF-1 overcomes the cell death signal induced by inhibition of MEK1 and MEK2 without an increase in MAP-kinase phosphorylation. These results suggest that inhibition of the Grb2/Ras/MAP-kinase pathway is unlikely to be effective in the treatment of oestrogen-responsive breast cancer unless treatment is combined with inhibition of IGF signal transduction.
IGFs may signal through the type I IGF receptor, through isoform A and, to a lesser extent, through isoform B of the insulin receptor [
9,
34,
49]. The receptor involved is of clinical importance because drugs such as figitumumab, cixutumumab, ganitumab and dalotuzumab are directed specifically against the type I IGF receptor [
16‐
19] whereas BMS-754807 and linisitinib inhibit both the type I IGF and insulin receptors [
20,
21]. Our data indicate that signalling through the type I IGF receptor is dominant in oestrogen-responsive breast cancer.
Conclusions
We have established a reliable model of caspase-dependent anoikis for oestrogen-responsive breast cancer cells. We demonstrate the importance of the intrinsic pathway in anoikis and that IGF-1 can reinstate anoikis resistance of unattached oestrogen-responsive breast cancer cells cultured in serum-free medium. IGF-1 activated both the PI3-kinase/Akt and Grb2/Ras/MAP-kinase pathways but our experiments with PI3-kinase, Akt and MEK inhibitors show that anoikis resistance is conferred through the PI3-kinase/Akt pathway. Although ERK1 and ERK2 are not important in IGF-dependent anoikis-resistance, IGF-1 is able to circumvent apoptosis induced by inhibition of MEK1 and MEK2 without any increase in MAP-kinase phosphorylation. The IGF-1 signal is transduced via the type I IGF receptor and incubation with type I IGF receptor specific antibody, figitumumab, induces anoikis of cells grown in serum. This is the first demonstration of the importance of IGFs and the type I IGF receptor in the resistance of oestrogen-responsive breast cancer cells to caspase-dependent anoikis. The importance of the type I IGF receptor is supported by the association of higher receptor expression with earlier relapse with distant metastases in oestrogen receptor-positive tumours, especially those of women with more aggressive disease as assessed either by presence of involved lymph nodes or high histological tumour grade. Successful targeting of the type I IGF receptor would abrogate effectively the IGF signal for cell survival. Collectively, our results support the concept that effective combinations of targeted drugs should include abrogation of the activity of the IGF signal transduction pathway.
Methods
Cell culture
Breast cancer cell lines: MCF-7, T-47D, ZR-75 and EFM-19 were obtained from the American Type Culture Collection (Manassas, VA) or DMCSZ and cultured routinely in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, Poole, United Kingdom), supplemented with 10 % foetal calf serum (FCS) and 1 μgml−1 insulin in a humidified incubator at 37 °C with 5 % CO2.
Anoikis assay
The non-ionic acid poly(2-hydroxyethyl methacrylate) (poly-HEMA; SIGMA) which inhibits matrix deposition and cell attachment [
26] was dissolved in 99 % ethanol at 10 mgml
−1. Twelve-well tissue culture plates were coated twice with 0.5 ml poly-HEMA solution, allowed to dry, washed with phosphate-buffered saline (PBS) and stored at 4 °C.
Cells were trypsinised, 40 × 10
4 cells added to 35-mm-diameter poly-HEMA-coated wells and cultured in maintenance medium or in serum-free, phenol red-free DMEM in the absence or presence of IGF-1 for different lengths of time. Cells were recovered, centrifuged, and lysed for protein analysis. Cells were incubated without and with 20 μM PI3-kinase inhibitor, LY294002, (Sigma), 100 nM Akt inhibitor, GSK 690693, (SYN│thesis med chem Pty Ltd., Cambridge, United Kingdom) or 6 μM MEK 1 and MEK2 inhibitor, UO126, (SIgma) for 30 min prior to addition of IGF-1. The type I IGF receptor inhibitory antibody figitumumab [
16] was from Pfizer Inc. (Tadworth, United Kingdom). Cells were centrifuged, and lysed for protein analysis.
Activation of caspase 3 was measured with the BD Pharmingen FITC-conjugated active caspase 3 apoptosis kit I (BD Biosciences, Oxford, United Kingdom). Cells were added to 16-mm-diameter poly-HEMA-coated wells at 40 × 104 cells/well in 2 ml of serum-free medium in the absence or presence of 50 ng/ml IGF-1 and incubated for 5 h. Cells were recovered, centrifuged, washed in PBS and, resuspended in 0.2 ml Cytofix/Cytoperm solution and incubated on ice for 20 min. Cells were then washed twice with 0.2 ml Perm/Wash solution, resuspended in 100 μl Perm/Wash solution with 8 μl and FITC-conjugated anti-activated caspase 3 antibody, protected from light and incubated for 30 min. Cells were rewashed, centrifuged and resuspended in 400–500 μl Perm/Wash solution. Fluorescence was measured with a BD FACScan flow cytometer. Excitation was at 488 nm and emission was measured at 530 ± 15 nm.
Knockdown of the type I IGF receptor
The sequence of the double-stranded short interfering RNAs (siRNA) designed to target the type I IGF receptor mRNA was 5’-CGACUAUCAGCAGCUGAAGTT-3’, and equivalent non-silencing scrambled sequence was 5’-UUCUCCGAACGUGUCACGUdTdT-3’ (Sigma). The siRNA duplex was mixed with RNA iMax (Invitrogen, Paisley, United Kingdom) in serum-free DMEM and incubated 30 min at room temperature. MCF-7 cells were trypsinised, resuspended in maintenance medium at a density of 25 × 104 cellsml−1, mixed with the transfection medium and added to a 25 cm2 flask. Cells were tested in anoikis assays or lysed for protein analysis, after incubation for 48 h or 96 h, respectively.
IGF-1 stimulated protein phosphorylation
Cells were added to 16-mm-diameter wells at a density of 15 × 10
4 cells/well for MCF-7 and ZR-75 cells, and 20 × 10
4 cells/well for EFM-19 cells. Cells were allowed to attach for 24-48 h and withdrawn from stimulatory factors in serum by culture for 48 h in growth factor-depleted medium, comprising phenol red-free DMEM and 10 % dextran-coated, charcoal-treated serum. Medium was changed daily. Cells were incubated with different concentrations of IGF-1 for 15 min, lysed and analysed by Western transfer [
12].
Western transfer analysis
Cells were lysed in radioimmunoprecipitate (RIPA) buffer which comprised 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 % NP-40 (v/v) and 0.25 % sodium deoxycholate (w/v, 1 μgml
−1 pepstatin, 1 μgml
−1 aprotinin, 1 μgml
- 1 leupeptin, 2 mM sodium orthovanadate, 2 mM sodium fluoride and 2 mM phenyl methyl sulphonyl fluoride. Protein concentrations were measured with a bicinchonic acid assay (Thermo Scientific, Loughborough, United Kingdom). Equal amounts of protein were separated by electrophoresis on denaturing 12 % polyacrylamide gels and transferred to a Westran 0.45 μm nitrocellulose membrane (VWR, Leicestershire, United Kingdom) [
50]. Membranes were incubated with specific antibodies against: cleaved poly-(ADP-ribose) polymerase (PARP) (#9541), type I IGF receptor (#3027), phosphorylated type I IGF receptor Tyr1135&1136 and insulin receptor Tyr1150&1151. (#3024), Akt (#9272), phosphorylated Akt Ser473 (#4060), FAK (#3285), phosphorylated FAK Tyr397 (#3283), ERK1 and ERK 2 (#9102), phosphorylated ERK 1 and ERK2 Thr 202 or Tyr 204 (#4370), Bad (#9292), phosphorylated Bad Ser136 (#4366), GSK3β (#9315), phosphorylated GSK3β Ser9 (#9323) (Cell Signaling Technologies, Hitchin, United Kingdom), IRS-1 (sc-7200), phosphorylated IRS-1 Tyr632 (sc-17196-R), and GAPDH (sc-25778) (Santa Cruz Biotechnology, Heidelberg, Germany). Membranes were incubated with horseradish peroxidase conjugated secondary antibodies followed by enhanced chemiluminescence with SuperSignal West Dura Substrate (Thermo Scientific) and exposure to SuperRX X-ray film (Fujifilm, Bedford, United Kingdom). The intensity of the protein bands was quantified by densitometry with Labworks 4.0 software (UVP Inc., Cambridge, United Kingdom).
Statistics
For the western transfer images, a representative example is shown. Data were normalized and expressed as a percentage of the maximum cleaved PARP or activated signal transduction protein detected. Results are expressed as means ± S.E.M. Differences between groups were tested by analysis of variance, paired or unpaired t-test; p < 0.05 was considered statistically significant. Experiments were replicated at least thrice. The association between type I IGF receptor expression and time to relapse with distant metastasis was analysed by the log rank test on data from The Cancer Genome Atlas.
Competing interests
The authors declare no competing interest.
Authors’ contributions
BCL conceived and undertook experiments and analysed data. FEBM conceived experiments. Both authors were involved in preparation of the paper. Both authors read and approved the final manuscript.