Background
Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with exposure to asbestos. Currently rates of MPM are rising and estimates indicate that the incidence of MPM will peak within the next 10–15 years in the western world, while in Japan the peak is predicted not to occur until 40 years from now [
1,
2]. Although the use of asbestos has been banned in many countries around the world, production of and the potentially hazardous exposure to asbestos is still present with locally high incidences of mesothelioma [
3]. Today a new man-made material, carbon nanotubes (CNTs), has arisen as a concern; CNTs may display ‘asbestos-like’ pathogenicity with mesothelioma induction potential [
4,
5]. The pharmacology of CNTs, including effects on immune responses and tissue accumulation, is therefore intensely investigated; “it is better to be safe than sorry” [
6].
MPM is an extremely difficult disease to treat, with a median overall survival ranging between 9 and 17 months, regardless of disease stage [
7,
8]. The combination of pemetrexed and cisplatin has been established as the current standard of care (SOC) but only 40% of treated patients show response to this therapy, with an overall median survival of 12.1 months [
9].
There is an urgent need for new targeted therapy for clinical management of MPM that can stop progression and stabilize the disease or ideally erase the tumor, either as add-on to SOC, to improve treatment efficacy and reduce chemoresistance and toxicity, or as monotherapy for patients whose performance status does not allow aggressive treatment.
Rapid advances in the understanding of cancer biology are leading to identification of new targets for cancer treatment and personalized therapy is rapidly becoming a reality with the aim of improving survival and quality of life for cancer patients [
10‐
12].
The expression of estrogen receptor beta (ERβ) in malignant pleural and peritoneal mesothelioma correlates with longer patient survival and is an independent prognostic factor [
13,
14]. Our group recently demonstrated that 70-80% of MPMs express ERβ, which acts as a tumor suppressor, inhibiting MPM cell proliferation and invasiveness [
13,
15].
ERβ is the second ER subtype identified in several human tissues traditionally believed to be ER negative [
16]. ERβ is expressed and demonstrated to exert anti-proliferative effects in different preclinical
in vitro and
in vivo models of human cancers, for example, breast, colon, prostate, lymphoma, and pleural mesothelioma [
13,
15,
17‐
25]. Moreover, ERβ is proposed to mediate the beneficial clinical effects in ERα negative breast cancer patients [
26], in malignant intraperitoneal mesothelioma [
27], and in the prevention of colon cancer in women on menopausal hormone therapy (MHT) [
28].
Drugs that selectively target ERβ might be safer than non-selective estrogens, which are associated with increased risk of breast, endometrial and ovarian cancer in women and the development of prostate cancer in men. These serious side effects of non-selective estrogens are mediated by the ERα subtype [
29‐
32]. Several synthetic and natural ERβ-selective compounds have been identified [
19,
33‐
36] that have shown promising anti-tumorigenic efficacy in preclinical cancer models [
37‐
43]. Therefore, drugs with selectivity for ERβ might prove promising in the the development of novel, targeted therapies for the clinical management of human cancers.
In the present study, we characterized the efficacy of KB9520, a selective ERβ agonist, to inhibit MPM cell growth in vitro and in vivo. Moreover, we investigated the possibility of an additive or synergistic effect between KB9520 and the SOC regimen (cisplatin/pemetrexed) for treatment of MPM.
Discussion
MPM is typically refractory to current treatment options using chemotherapy. In a first line setting, pemetrexed in combination with cisplatin has been accepted as an almost universal standard. In the second line setting, various chemotherapy agents have been used, either as monotherapy or as part of polytherapy, but none has been successfully validated.
A recently published review, based on results of a meta-analysis of unresectable MPM, suggests that response rate and survival are greater for combination therapy than for single-agent regimens, and that platinum-containing regimens have greater efficacy than non-platinum-containing combinations, confirming that platinum-based chemotherapy remains the most effective treatment for patients with MPM [
45].
Our group has recently demonstrated that ERβ exerts a key role as a tumor-suppressor gene in MPM [
13,
15] and its activation may also explain the gender difference clinically observed for the prognosis of MPM.
In this study we report that activating ERβ with the highly selective ligand KB9520 resulted in a concentration and time-dependent inhibition of REN malignant mesothelioma cell growth
in vitro. Both KB9520 and E2 displayed bell-shaped responses with growth inhibition at low doses and opposite effect at high doses. Bell-shaped responses to hormones are not unusual phenomena; “hormesis” is a biphasic dose response phenomenon characterized by a low dose stressful stimulation and a high dose adaptive response (inhibition) that increases the resistance of the cell to evoked stress [
46,
47]. Both ER subtypes, α and β, exert genomic and no-genomic effects which may explain the observed biphasic responses of KB9520 and E2. Other plausible explanation could be the cyclic on/off process of ER on promoters [
48,
49] leading to degradation of the ERβ protein.
Although KB9520 and E2 had similar growth inhibitory effect on the REN cells there was a difference in time to maximal effect, 24 hours for KB9520 and 72 hours for E2. This time to effect difference between KB9520 and E2 may depend on the different ERβ conformational change/s induced by KB9520 and E2, respectively, which in turn may have an effect on co-regulatory protein interactions including type of co-regulatory complex, stoichiometry and kinetics.
Evaluation of the anti-proliferative effect of KB9520 on a collection of human malignant mesothelioma cell lines showed a strict dependence on ERβ expression and that the growth inhibitory efficacy was related to the level of ERβ expressed. Lack of anti-proliferative effect of KB9520 in the ERβ positive non-malignant mesothelium derived MET5A cells is difficult to understand and needs further investigation. However, the difference in the regulatory mechanisms/signaling pathways for cell growth, survival and metabolism between non-malignant and malignant cells may somehow influence the activity of ERβ and its susceptibility to respond to an ERβ agonist. Differences in post-translational modification/s of ERβ and/or cell compartment localization are plausible explanations for activity or no activity of ERβ in malignant compared to non-malignant cells. Of note though, the non-malignant MET5A cells did respond to KB9520 when stressed by cisplatin.
The enhanced growth inhibitory effect of cisplatin/pemetrexed in combination with KB9520 in REN cells in vitro translated to synergistic anti-tumorigenic activity in vivo. Treatment with KB9520 in combination with cisplatin/pemetrexed in vivo had greater efficacy than either treatment alone and caused a significantly reduced tumor load compared to vehicle treated animals at the end of the treatment period. Moreover, the triple combination shrunk the tumor volume even below the tumor volume at the start of treatment.
Also in a second human malignant mesothelioma cell line, MMP [
13], KB9520 in complex with cisplatin resulted in synergistic growth inhibition compared to KB9520 or cisplatin alone
in vitro (data not shown). Moreover, similar to the effect in REN cells, the combination of KB9520 and cisplatin in the MMP cells decreased the level of phosphorylated AKT and increased the levels of cleaved PARP1 (data not shown).
That a brief exposure of malignant mesothelioma cells to KB9520 (2 hours) elicited a stronger growth inhibitory effect in vitro compared to continuous exposure is suggestive of a hit-and-run mechanism. The long-lived biological activity of KB9520 (≥24 hours) together with a hit-and-run type of mechanism adds to our understanding of the in vivo tumor inhibitory efficacy of KB9520 despite its short plasma half-life of approximately 1 hour in mice.
The PI3K/AKT signaling pathway is aberrantly active and plays a critical role in cell cycle progression and cell survival in human MPM including the sensitivity to cisplatin [
50]. We have previously demonstrated a role of AKT activation in MPM cell response to cisplatin [
44]. Here we show that KB9520 significantly reduced pAKT levels both
in vitro and
in vivo (data not shown), which, at least in part, may explain the observed sensitization to cisplatin cytotoxicity. The observed reduction in AKT phosphorylation warrant further investigation; it could be due to either modulation of the rate of both protein kinase and phosphatase activities or to AKT post-translational modification that could affect its localization and/or activation status.
The order of drug administration may sometimes be very important for optimal therapeutic efficacy [
51,
52]. Exposure of REN cells to KB9520 prior to cisplatin resulted in synergistic inhibition of malignant mesothelioma cell proliferation and survival whereas the reverse order of drug exposure did not even result in additive efficacy. Furthermore, KB9520 preconditioned malignant mesothelioma cells to low-concentration cisplatin cytotoxicity; combination of KB9520 with 20 μM cisplatin was as efficacious as 100 μM cisplatin alone. Thus, these data imply that KB9520, through ERβ, acts as a chemosensitizer increasing cisplatin cytotoxicity in human malignant mesothelioma cells.
Chemotherapy and in particular cisplatin is widely used in the treatment of various human solid tumors. However, cisplatin is associated with serious toxicity, which limits its use; the majority of patients diagnosed with MPM are older than 65 years and their health condition may therefore not allow the standard chemo dosing regimen of cisplatin/pemetrexed. To investigate the cytotoxic effect of the cisplatin/KB9520 combination in non-malignant mesothelial cells we treated the mesothelium-derived MET5A cells with various concentrations of cisplatin in the presence or absence of KB9520. In contrast to the effect in the malignant REN cells KB9520 diminished the toxicity of cisplatin in the MET5A cells, in part explained by the reduced PARP1 cleavage and increased pAKT levels. It has been described that ER can increase PI3K/AKT activity by interaction with the p85 subunit of PI3K [
53,
54]. However, if that explains the increased pAKT levels in the MET5A cells needs to be explored in more detail.
Methods
Reagents and antibodies
The monoclonal antibodies specific for α-Tubulin, PARP1 and the polyclonal antibody specific for ERβ were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-AKT (pSer473) was from Cell Signaling Technology (Beverly, MA, USA), anti-mouse and anti-rabbit IgG peroxidase conjugated antibodies and chemical reagents were from Sigma-Aldrich (St Louis, MO, USA). ECL was from Amersham Pharmacia Biotech (Uppsala, Sweden). Nitrocellulose membranes and protein assay kit were from Bio-Rad (Hercules, CA, USA). Culture media, sera, antibiotics and LipofectAMINE transfection reagent were from Invitrogen (Carlsbad, CA, USA). The ERβ selective agonist KB9520 (see Additional file
1: Figure S1) was designed and synthesized by Karo Bio (Huddinge, Sweden). (KB9520 has been described previously [
36,
41‐
43]. The compound can be obtained following contact with Karo Bio AB [stefan.nilsson@karobio.se] and after signing of a Material Transfer Agreement together with a detailed protocol of planned study. A fee covering the cost of compound synthesis will be charged).
Cell cultures and transfection
The epithelioid MPM derived REN cell line, used as the principal experimental model in this investigation, was isolated, characterized and kindly provided by Dr. Albelda S.M. (University of Pennsylvania, Philadelphia; PA, USA). Cells were characterized by BMR Genomics s.r.l. using the PowerPlex 18D System kit. The biphasic MSTO-211H and the mesothelial MET5A cell lines were obtained from the Istituto Scientifico Tumori (IST) Cell-bank, Genoa, Italy; the MMB cell line derived from pleural effusions of patients with MPM and stabilized in culture [
55]; the H2596 cell line produced by Dr. H. I. Pass from surgical specimens derived from patients with resected MPM [
56] were kindly provided by Dr. W. Thomas (RCSI, Dublin, IRL) in 2011. Cells were grown in RPMI medium supplemented with 10% FBS, 100 μg/ml streptomycin and 10 μg/ml penicillin at 37°C in a humidified environment containing 5% CO2. Mycoplasma infection was excluded by the use of Mycoplasma PlusTM PCR Primer Set kit from Stratagene (La Jolla, CA, USA). Cells grown to 80% confluence in tissue culture dishes were transiently transfected with the pCNX2 plasmid expressing human wild type ERβ (Addgene, Cambridge, MA, USA) using LipofectAMINE reagent as described by the manufacturer.
Gene silencing was achieved using an ERβ-specific shRNA lentiviral plasmid (pLKO.1-puro) by Sigma (St Louis, MO, USA) or specific siRNAs by Qiagen (Hilden, Germany).
Proliferation assays
Cells were seeded at a density of 10×104 cells/well in 6-well plates in RPMI medium supplemented with 10% FBS, 100 μg/ml streptomycin and 10 μg/ml penicillin and incubated over-night at 37°C in a humidified environment containing 5% CO2 to allow adherence. Following treatment cells were trypsinized and stained with Trypan blue. The number of cells considered viable (unstained cells) was counted in a Bürker haemocytometer within 5 minutes after staining.
Wash-off experiments
Cell cultures were pretreated with KB9520 for 1–16 hours (depending on experiment) followed by wash-off and then replenished with normal growth medium ± cisplatin, pemetrexed or cisplatin/pemetrexed. Total incubation time was 24–72 hours (depending on experiment). Control cultures were maintained in normal growth medium without added drug. The number of viable cells was determined as described. For details of each experiment see figure legends.
Add-on experiments
In the add-on experiments the second drug was added directly to the cell culture medium without wash-off of the first drug. Total incubation time was 24 hours. Control cultures were maintained in normal growth medium without added drug. The number of viable cells was determined as described.
Cell lysis and immunoblot
Cells were extracted with 1% NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris–HCl pH 8.5 mM EDTA, 10 mM NaF, 10 mM Na4P2O7, 0.4 mM Na3VO4) with freshly added protease inhibitors (10 μg/ml leupeptin, 4 μg/ml pepstatin and 0.1 Unit/ml aprotinin). Lysates were centrifuged at 13.000 × g for 10 minutes at 4°C and the supernatants were collected and assayed for protein concentration with the Bio-Rad protein assay method.
Proteins were separated by SDS-PAGE under reducing conditions. Following SDS-PAGE, proteins were transferred to nitrocellulose, reacted with specific antibodies and then detected with peroxidase-conjugate secondary antibodies and chemiluminescent ECL reagent. Densitometric analysis was performed using the GS 250 Molecular Image (Bio-Rad).
Cell cycle analysis
For cell cycle/apoptosis analysis, 5 × 105 cells were seeded in tissue culture plates and treated with 10 nM KB9520, 100 μM cisplatin or the combination of the two drugs for 24 hours at 37°C in a 5% CO2 atmosphere. After incubation, detached and suspended cells were harvested in complete RPMI and centrifuged at 500 × g for 10 minutes. Pellets were washed with PBS, fixed in ice-cold 75% ethanol at 4°C, treated with 100 mg/mL RNAse A for 1 hour at 37°C, stained with 25 μg/mL propidium iodide and finally analyzed by using a flow cytometer FACS (Becton Dickinson, San Jose, CA, USA) and Modfit software (Verity Software House, Topsham, ME, USA).
In vivo experiments
Animals
CD1 nude mice (males, 6 weeks old; Charles River, Calco, Italy) received intra-peritoneal (i.p.) injections of 2×106 luciferase transduced REN cells in 0.5 mL of RPMI medium. After anesthetization and i.p. injections of 0.3 mL of 15 mg/mL D-luciferin, tumor dimension and localization of luminescent cells was monitored using the In Vivo Imaging System (IVIS®) system 100 series (Xenogen Corporation, Hopkinton, MA, USA). Regions of interest were identified around the tumor sites and were quantified as total photon counts using Living Image software (Xenogen Corporation). The values of tumor sizes were obtained by subtracting luminescence signals of each weekly measurement by the average of all animals within a treatment group on the 15th day after inoculation (day when treatment started). To evaluate treatment toxicity, mice were weighed at the start and end of treatments. Mice were killed and necropsied after 20 days of treatment. In vivo experiments were approved by Istituto Scientifico Tumori (Genoa, Italy) ethical committee and conform to the relevant regulatory standards. Mice were maintained and handled under aseptic conditions, and were allowed access to food and water ad libitum.
Drug administration
An elapse of 15 days was allowed for the formation of detectable tumor nodules, assessed by IVIS® imaging. Mice were then weighed and stratified into treatment groups of ten animals. Treatment protocols were done from the 15th day to the 35th day, and mice were analyzed weekly by IVIS® imaging to assess tumor growth. One dose of KB9520 was used (10 mg/kg/day). KB9520 was dissolved in the vehicle (5% DMSO/40% PEG 400/55% water) and administrated once daily (days 15–35) by sub-cutaneous administration. 5 mg/kg cisplatin solution (Ebewe Italia srl, Roma, Italy) was administrated subcutaneously at day 18 and 25, respectively, and 150 mg/kg pemetrexed (dissolved in isotonic saline) (Eli Lilly, Houten, Nederland) was injected subcutaneously at days 19–23 and 26–30, respectively. Untreated animals were dosed with empty vehicle. At day 35 mice from the four groups were euthanized and necropsied. Tumors growing in the peritoneum were excised, and one part of the tumor tissues was immediately frozen and stored at −80°C for subsequent analysis.
Statistical analysis
Statistical evaluation of the differential analysis was performed by one way ANOVA and Student’s t-test. The threshold for statistical significance was set at p ≤ 0.05. The statistical analysis of
in vivo experiments was done by using R [
57]. To compare different groups we used the non-parametric Kruskal-Wallis test; if differences were found significant (p ≤ 0.05) we subsequently applied the Wilkoxon rank sum test to do the pair wise comparisons. To compare tumor growth curves we used the StatMod package.
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Competing interests
SN is an employee at Karo Bio AB. The remaining authors declare that they have no competing interests.
Authors’ contributions
GP and AGM carried out cellular and molecular studies and made substantial contributions to analysis and interpretation of data, AD and MC performed in vivo studies. MR conducted statistical analysis. SN and LM participated in the design and coordination of the study and drafted the manuscript. All authors have read and approved the final manuscript.