Background
In approximately 15% of invasive breast cancers, estrogen receptor (ER) and progesterone receptor expression is low or undetectable, and human epidermal growth factor (EGF) receptor-2 (HER2) is not overexpressed. These triple-negative breast cancers (TNBC) carry a poorer prognosis than other breast cancer types [
1,
2]. A majority of TNBC falls into the basal-like molecular classification, and conversely, most basal-like cancers are triple-negative [
3,
4]. Among six defined molecular subtypes of TNBC [
5], those identified as basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), and mesenchymal are overwhelmingly classified as having basal-like molecular signatures [
3,
4]. Since TNBC is not susceptible to ER- or HER2-targeted therapies, these cancers are generally treated with adjuvant, and sometimes neoadjuvant, chemotherapy, often with substantial survival benefit [
4]. Adjuvant radiotherapy may also improve survival in some patient groups [
6]. However, the risk of relapse within the first 3–4 years is relatively high [
2]. In attempts to overcome this, numerous targeted therapies have been trialed, alone or in combination with chemotherapy [
4], but the need remains for new treatment options for women with TNBC.
The EGF receptor (EGFR) is often highly expressed in TNBC tumors, and is prognostic for worse disease-free survival [
7], although EGFR mutation is uncommon [
8]. Single-agent blockade of EGFR signaling has been disappointing in TNBC patients [
9,
10], suggesting that combination therapies may be needed. The growth-regulatory protein, insulin-like growth factor binding protein-3 (IGFBP-3), potentiates EGFR signaling in MCF-10A mammary epithelial cells – which are triple-negative, basal B [
11] – and several TNBC cell lines [
12,
13]. This potentiation occurs via activation of sphingosine kinase 1 (SphK1) and generation of sphingosine-1-phosphate (S1P) [
12,
13], a lipid mediator of oncogenesis [
14]. Direct binding of IGFBP-3 to EGFR, and their nuclear colocalization, in TNBC cells are enhanced in response to DNA-damaging chemotherapy [
15]. ER-negative breast tumors also typically have higher expression of IGFBP-3 mRNA [
16] and protein [
17] than ER-positive tumors, and in women with breast cancer of the basal-like subtype, high IGFBP-3 is significantly associated with poorer recurrence-free survival [
18]. Finally, SphK1 is also more highly expressed in ER-negative/TNBCs than in ER-positive breast cancer, and is significantly related to poorer disease-free survival [
19].
Since IGFBP-3, which is abundant in the circulation as well as the cellular environment, is challenging to target, the strong associations among IGFBP-3, EGFR and SphK1 prompted us to evaluate co-targeting of the IGFBP-3 mediators EGFR and SphK1 in TNBC cell lines in vitro and in xenograft tumors. The SphK1 inhibitor, SKI-II, showed a powerful combined inhibitory effect with the EGFR tyrosine kinase inhibitor (EGFR-TKI) gefitinib (Iressa, AstraZeneca, Cambridge, UK), with significant inhibitory action under conditions where neither single agent was significantly inhibitory [
13]. To translate this finding clinically it would be advantageous to use drugs in current clinical practice, with known toxicity and pharmacokinetics. FTY720 (Fingolimod, Novartis, Basel, Switzerland) is a structural analogue of sphingosine with S1P receptor 1 (S1P
1) modulatory activity, that has clinical benefit in relapsing-remitting multiple sclerosis owing to its ability to inhibit T-cell migration from lymph nodes [
20]. Among a range of other activities, FTY720 is also an inhibitor of SphK1 and has been proposed as a potential cancer therapy [
21‐
24].
In this study, we demonstrate a powerful synergistic inhibition of TNBC cell growth by treatment with a combination of EGFR-TKI and FTY720, and show that, despite its potential immunomodulatory effect, FTY720 is effective in extending survival in both immune-deficient xenograft and immune-competent syngeneic mouse models.
Methods
Reagents, drugs and cell lines
Human TNBC cell lines, and the murine 4T1 mammary carcinoma cell line, were obtained from ATCC, Manassas, VA, USA and maintained in RPMI 1640 medium containing 5% FBS and 10 μg/mL bovine insulin under standard conditions. Identity of Hs578T cells, which were obtained from ATCC in 2001, was confirmed by short-tandem repeat profiling by CellBank Australia (Westmead, NSW, Australia) in December 2012. Cryopreserved stocks of other cell lines (purchased in 2010 from ATCC) were established within 1 month of receipt, and fresh cultures for use in experiments were established from these stocks every 2-3 months. All cell lines tested negative for mycoplasma contamination. Stable downregulation of IGFBP-3 was achieved by transfecting 2 × 10
6 cells with SureSilencing shRNA or negative control plasmids according to the manufacturer’s instructions (Qiagen, Melbourne, VIC, Australia) and selection using hygromycin B. Cell lysates and media were collected post-selection to validate knockdown efficiency by qRT-PCR, radioimmunoassay and immunoblotting as previously described [
13].
Gefitinib (Iressa; ZD1839, AstraZeneca) and FTY720 (Fingolimod; Gilenya, Novartis) were obtained from MedChem Express, Monmouth Junction, NJ, USA. Desmethyl erlotinib (OSI-420) was obtained from AdooQ BioScience, Irvine, CA, USA.
Cell growth studies
Cell proliferation was studied using the IncuCyte live-cell imager (Essen BioScience, Ann Arbor, MI, USA) as previously described [
13]. Cells (1 × 10
3/well for 4T1, 2 × 10
3/well for Hs578T, 4 × 10
3/well for HCC70, HCC1806, MDA-MB-231 and MDA-MB-436, and 8 × 10
3 cells/well for MDA-MB-468) were dispensed into 96-well plates in complete medium and incubated overnight before changing to fresh medium containing 5% FBS and inhibitors. Plates were transferred to the IncuCyte, and incubations continued over 72–136 h, depending on the cell line, with images collected every 3 h.
Mouse models of TNBC
All animal procedures were approved by the institutional Animal Ethics Committee (Protocols RESP/14/280 and RESP/15/103). Female BALB/c nude mice, 6–7 weeks old, were obtained from Animal Resource Centre, Murdoch, Western Australia, and were acclimatized under local conditions before use as hosts for tumor xenografts at 8 weeks. Wildtype BALB/c mice were obtained from the institutional animal facility. For all tumors, cells were mixed with 50 μL Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and injected in 150 μL total volume into the 4th left mammary fat pad. Tumor volumes were measured three times weekly and mice were weighed weekly. All treatments were administered intraperitoneally (i.p.) three times weekly.
MDA-MB-468 tumors
5 × 106 MDA-MB-468 human mammary carcinoma cells were implanted in nude mice. When tumors reached 100 mm3 (length × width2/2), mice were randomized into groups of five mice for treatment. Protocol 1: FTY720 at 3 mg/kg (or vehicle) and gefitinib at 50 mg/kg (or vehicle). All mice were terminated on day 25 of treatment, the day when the largest tumor reached 500 mm3. Protocol 2: FTY720 at 5 mg/kg (or vehicle) and gefitinib at 25 or 50 mg/kg (or vehicle). Each mouse was terminated when its tumor reached 500 mm3, or at treatment day 40.
HCC1806 and HCC70 tumors
5 × 106 HCC1806 or HCC70 human mammary carcinoma cells were implanted in nude mice. When tumors reached 200 mm3 (100 mm3 for HCC70), mice were randomized into four groups of ten mice for treatment: FTY720 at 5 mg/kg and gefitinib at 25 mg/kg (50 mg/kg for HCC70). Each mouse was terminated when its tumor reached 1000 mm3 or at 12 weeks after injecting cells.
4T1 tumors
1 × 105 4T1 murine mammary carcinoma cells were implanted into BALB/c nude or BALB/c wild-type mice. When tumors reached 100 mm3, mice were randomized into four groups of ten mice for treatment: FTY720 at 5 mg/kg and gefitinib at 50 mg/kg. Each mouse was terminated when its tumor reached 1000 mm3.
Analytical methods – cell culture
IGFBP3, SPHK1 and
CD44 mRNA expression was measured, in duplicate, on duplicate RNA extracts by qRT-PCR as previously described [
13], using the following Taqman probes (Applied Biosystems, Foster City, CA, USA): IGFBP-3: Hs00181211_m1; SPHK1: Hs00184211_m1; CD44: Hs01075864_m1; and HMBS (reference gene): Hs00609297_m1. IGFBP-3 concentrations in cell-conditioned media were measured by in-house radioimmunoassay [
13]. Western blotting was performed as described previously [
13] using antibodies from Cell Signaling Technology (Beverly, MA, USA): total EGFR (#2232, 1:1,500), pEGFR (Tyr1068) (#2234, 1:1,500), HER2/ErbB2 (#2242, 1:1,000), type 1 IGF receptor (IGF1R, #3027, 1:1,000), p53 (#9282, 1:1,000), p63 (#4892, 1:1,000), CD44 (#3570, 1:1,500), vimentin (#3390, 1:1,000), E-cadherin (#3195, 1:1,000). SphK1 antibody (ab16491, 1:1,000) was from Abcam (Walnut, CA, USA) and α-tubulin antibody (T9026, 1:10,000) from Sigma-Aldrich, St Louis, MO, USA. Rabbit antihuman IGFBP-3 antiserum R-100 was raised in-house. Secondary antibodies were from Pierce Biotechnology (Rockford, IL, USA).
Analytical methods – tissue sections
Tumor samples were fixed in 10% phosphate-buffered formalin and embedded in paraffin. Four-micron sections were incubated with antibodies against Ki67 (ab66155, 1:600, Abcam, Melbourne, VIC, Australia), cleaved caspase-3 (Asp175) (#9661, 1:200, Cell Signaling), pEGFR (Tyr1068) (#2234, 1:300, Cell Signaling), SphK1 (#AP7237c, 1:400, Abgent, San Diego, CA, USA), CD44 (156-3C11, mouse mAb #3570, 1:200, Cell Signaling), IGFBP-3 (in-house antiserum R-100, 1:2000), or CD3 (ab16044, 1 μg/ml, Abcam) and isotyped-matched IgG antibodies. Immunodetection used the Dako EnVision + System-HRP labeled polymer detection kit (Dako, Carpinteria, CA, USA) with visualization using ImmPACT NovaRED Peroxidase (HRP) Substrate (# SK-4805, Vector Laboratories, Burlingame, CA, USA), and counterstaining by Mayer’s hematoxylin and Scott’s bluing solution. After mounting, sections were viewed by light microscope (Eclipse 80i, Nikon, Tokyo, Japan) and evaluated. For each antibody, all immunohistochemistry (IHC) was performed in a single assay to exclude between-run variability. Markers were evaluated by semi-quantitative scoring from the intact cell surface area from the five highest staining areas at × 10 magnification for each slide.
IHC staining of Ki67, cleaved caspase-3, IGFBP-3 and CD44 were scored as percentage of positive cells. Specific IGFBP-3 staining was mostly in the nucleus, and only nuclear staining was scored. For cleaved caspase-3 scoring, central necrotic parts of tumors were excluded. SphK1 and pEGFR were evaluated by both the percentage of target cells stained, and the staining intensity. The intensity was scored as 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong). The final score, ranging from 0–300, was obtained by multiplying the scores for intensity by the percentage of positive cells.
Data analysis and statistics
CompuSyn v1.0 software (ComboSyn Inc., Paramus, NJ, USA) was used to calculate the Chou-Talalay Combination Index (CI), where CI < 1indicates synergism and CI > 1 indicates antagonism [
25]. All other statistical analyses were performed using SPSS v.22 for Mac (IBM Corp, Armonk, NY, USA). Effects of drug treatment on tumor IHC staining scores were calculated using 1-factor ANOVA followed by Tukey’s post hoc test. Pearson’s correlations among IHC staining scores are reported with two-tailed
P values.
Discussion
Combinations of receptor tyrosine kinase inhibitors have previously been tested in breast cancer cell lines, including TNBC cells [
35]. In the present study, we have evaluated the novel combination of tyrosine kinase (EGFR) inhibition with lipid kinase (SphK1) inhibition for its ability to block IGFBP-3-dependent proliferative signaling in TNBC, based on the observations that: (i) IGFBP-3 is abundant in many TNBC tumors, and prognostic for poor patient survival, (ii) endogenous IGFBP-3 potentiates EGFR signaling and EGFR-dependent cell proliferation, (iii) IGFBP-3 activates the oncogenic lipid kinase SphK1, and (iv) the effects of IGFBP-3 on EGFR-dependent proliferation are blocked by sphingosine kinase inhibition or downregulation. IGFBP-3 is abundant in the circulation where it is the predominant transport protein for the growth factors, IGF-1 and IGF-2 [
36]. Circulating IGFBP-3 is mainly found in ternary complexes with the acid-labile subunit (ALS), with limited egress from the vasculature. Thus tissue effects of IGFBP-3 may largely be attributable to local (tissue and tumor) production. In breast cancer tissue, IGFBP-3 is predominantly associated with ER-negative tumors, with much lower expression in ER-positive breast cancer [
16]. Since modulating the tissue (autocrine or paracrine) effects of IGFBP-3 by blocking the abundant circulating protein would be therapeutically challenging, we co-targeted two kinases that mediate at least some of its tissue effects in TNBC, EGFR and SphK1.
Whereas EGFR kinase inhibitors, such as gefitinib and erlotinib, are in routine clinical use, the development of SphK1 inhibitors is less advanced [
37]. FTY720, used clinically for immunomodulation in relapsing-remitting multiple sclerosis, also shows efficacy in preclinical cancer models [
21,
24]. It has multiple molecular targets, of which the downregulation of S1P
1 receptors, leading to inhibition of T-lymphocyte mobilization from lymphoid tissue, is perhaps the best characterized [
20]. FTY720 also has well-characterized SphK1 inhibitory activity, for which it was selected in this study. However, since we have shown that S1P
1 inhibition or downregulation prevented the potentiation of EGFR signaling by IGFBP-3 in the triple-negative, nonmalignant mammary cell line MCF-10A [
12], S1P
1 downregulation as well as SphK1 inhibition might contribute to FTY720 efficacy in our studies.
Gefitinib-FTY720 combinations were profoundly growth-inhibitory to a variety of TNBC cell lines in vitro, under conditions where neither drug alone was very inhibitory. At optimized drug combinations, strongly synergistic effects were observed. Of the representative TNBC cell lines tested, this strong combination effect was seen in every case, with no distinction among the four TNBC subclasses tested: BL1, BL2, mesenchymal or MSL, while the murine TNBC cell line 4 T1 showed a similar synergistic response. Evaluated in a single cell line (HCC1806), the EGFR kinase inhibitor erlotinib showed somewhat less synergy than gefitinib, with slightly higher doses needed for the same inhibitory effect. Although gefitinib is typically used clinically at a higher dose than erlotinib, owing to different pharmacokinetics [
38], a higher IC
50 value (lower efficacy) in vitro for erlotinib compared to gefitinib has been reported in several TNBC cell lines [
39].
Both drug targets, EGFR and SphK1, are activated by a wide range of cell effectors, and it is clear that neither EGFR inhibitors nor SphK1 inhibitors uniquely target IGFBP-3 signaling. Yet the central role of IGFBP-3 in activating these pathways in TNBC is emphasized by the observation that stable downregulation of IGFBP-3 in Hs578T cells abolished, and in HCC1806 cells diminished, the synergistic interaction between the two drugs. This reinforces that tumor IGFBP-3 expression is involved in driving EGFR-SphK1-dependent proliferation in TNBC cells, and should be evaluated as a possible biomarker of efficacy of the inhibitory drug combination. Interestingly, nuclear IGFBP-3 abundance showed highly significant downregulation by the inhibitor combination. IGFBP-3 has well-documented roles in the nucleus [
40], and nuclear IGFBP-3 has been reported as a strongly negative prognostic indicator in prostate cancer [
41]. Although the precise mechanism of nuclear IGFBP-3 downregulation in response to gefitinib-FTY720 co-treatment is unknown, this effect would be expected to contribute to the antitumor actions of the combination treatment.
The transmembrane cell adhesion molecule, CD44, is known to promote tumorigenic signaling in breast cancer. It exists in a number of splice variant isoforms, of which the ~80-kDa form is known as standard CD44, lacking the extracellular-domain inserts found in larger variants [
27]. CD44 is recognized as a cancer stem cell marker and is enriched in basal-like breast cancers [
42]. Its high expression is associated with poor disease-free survival in women with TNBC [
30]. Because IGFBP-3 has been identified as a positive regulator of CD44-high cells [
26], and the IGFBP-3 downstream mediators, EGFR and SphK1, are also known to regulate CD44 [
28,
29], we examined CD44 abundance in TNBC cells treated with the drug combination. In three TNBC cell lines, CD44 downregulation by gefitinib was potentiated by the addition of FTY720, suggesting CD44 as a plausible intermediate in a growth-stimulatory signaling cascade that is inhibited by dual EGFR-SphK1 blockade. In vivo, however, no changes in total tumor CD44 were seen among treatment groups by IHC. Since only the ~80-kDa CD44 isoform was measured by western blot in cell lysates, but most or all of the multiple variant forms would be detected by IHC, it is possible that changes in regulated isoforms might be masked by other abundant isoforms comprising total tissue CD44. Despite the lack of posttreatment downregulation seen in vivo, it will be important to determine in prospective studies whether high CD44 expression, already identified as a prognostic indicator in TNBC, might have value in predicting responsiveness to the combination therapy.
When tested in xenograft models in nude mice, the gefitinib-FTY720 combination significantly inhibited the growth of MDA-MB-468 and HCC1806 tumors, and extended the survival of mice bearing these tumors, compared to vehicle treatment or either monotherapy. In contrast, the survival of mice bearing HCC70 tumors was not significantly extended by the combination treatment. Since HCC70 and HCC1806 cells are comparable in IGFBP-3, EGFR and SphK1 expression, yet the treatment was most effective in HCC1806 tumors and least effective in HCC70 tumors (despite using the higher gefitinib dose of 50 mg/kg), additional factors may regulate treatment efficacy. Our cell proliferation studies indicated that, of the three cell lines, gefitinib sensitivity was highest in HCC1806 and lowest in HCC70 (data not shown), reflecting the effectiveness of the treatments in vivo, and suggesting that an even higher gefitinib dose may have been more effective in treating HCC70 tumors. Since initial trials in MDA-MB-468 tumors showed no difference between gefitinib at 25 or 50 mg/kg, three times weekly, subsequent xenograft tumors, HCC1806 and HCC70, were treated with gefitinib at 25 mg/kg. Other mouse studies have used doses up to 150 mg/kg/day orally for 5 days per week [
43,
44]; thus our dose of 25 mg/kg, chosen to demonstrate synergy with FTY720, was relatively low.
FTY720 was used at 3 or 5 mg/kg, three times weekly, in our combinations. This is also conservative for comparable animal studies, since other xenograft tumor studies have used doses up to 10 mg/kg every 2 days [
45,
46]. FTY720 has not been trialed in human cancer patients, but compared to the standard immunomodulatory dose of 0.5 mg/day for patients with multiple sclerosis [
32] – less than 0.01 mg/kg – doses used in cancer xenograft studies are extremely high. It is unclear why mice show such marked insensitivity to this drug compared to humans. Nevertheless, in the context of a possible clinical trial of the gefitinib-FTY720 combination in breast cancer patients, we investigated whether a FTY720 dose with antitumor efficacy in mice (in combination with gefitinib) would compromise the immune system. FTY720 inhibits T cell mobilization from lymphoid tissues [
20], which in some patients can result in lymphopenia [
32,
47]. If this depletes tumor-resident lymphocytes it could potentially be deleterious to breast cancer patients undergoing therapy, since a high tumor-associated lymphocyte count is a positive predictor of treatment response [
48].
Since this could not be examined in nude mice which lack mature T lymphocytes, we grew the murine mammary carcinoma cell line, 4T1, in the syngeneic mouse strain BALB/c, allowing us to compare the gefitinib-FTY720 combination in immune-deficient (nude) versus immune-competent (wild-type) mice. The hypothesis was that, since tumor-associated T cells are associated with improved treatment response, we would see greater drug efficacy in the immune-competent mice only if their immune system was not compromised by FTY720. Our observation of a highly significant extension of survival by the gefitinib-FTY720 combination in wild-type mice, compared to a nonsignificant effect in nude mice, suggests that FTY720 at a therapeutically active dose (in combination with gefitinib) did not prevent the enhancing effect of tumor lymphocytes on drug efficacy. A previous study suggested that, although FTY720 impairs T cell trafficking from lymphoid tissues, it does not appear to compromise the beneficial activity of intratumoral T cells [
49]. However, in a limited sample set, we saw a substantial decrease in tumor CD3 upon FTY720 treatment. Gefitinib therapy in patients has also been associated with lymphopenia [
50]. Surprisingly, in tumors treated with the gefitinib-FTY720 combination, the decline in tumor CD3 was greatly alleviated. While the mechanism of this combination effect remains to be determined, this suggests that FTY720, in combination with gefitinib, could be explored at its approved immunomodulatory dose of 0.5 mg/day, for efficacy in women with TNBC.
Immunohistochemical analysis of tumor tissues indicated that in mice bearing either MDA-MB-468 or HCC1806 xenograft tumors, the gefitinib-FTY720 combination was significantly more antiproliferative (decreased Ki67) and more pro-apoptotic (increased cleaved caspase-3) than either drug alone. This supports the cell culture data as well as the in vivo tumor growth and mouse survival data. Gefitinib shows strong selectivity for EGFR kinase activity, although there is also weak cross-reactivity with HER2 [
51]. In these triple-negative breast tumors the effect is likely to be entirely attributable to EGFR inhibition, and was reflected by significant inhibition of pEGFR by gefitinib treatment in HCC1806 tumor tissue, although the inhibition by gefitinib alone was not significant in MDA-MB-468 tumors. FTY720 treatment alone did not decrease pEGFR in either tumor type, but strongly enhanced the inhibitory effect of gefitinib. This potentiating effect of FTY720 on EGFR kinase inhibition has not previously been reported, although a combination effect of FTY720 (used as an S1P receptor antagonist) and the EGFR monoclonal cetuximab has been reported in colorectal cancer cells and xenograft tumors [
52].