Specific effects of bortezomib against experimental malignant pleural effusion: a preclinical study
© Psallidas et al; licensee BioMed Central Ltd. 2010
Received: 11 December 2009
Accepted: 10 March 2010
Published: 10 March 2010
We have previously shown that nuclear factor (NF)-κ B activation of mouse Lewis lung carcinoma (LLC) specifically promotes the induction of malignant pleural effusions (MPE) by these cells. In the present studies we hypothesized that treatment of immunocompetent mice with bortezomib tailored to inhibit cancer cell NF-κ B activation and not proliferation specifically inhibits MPE formation by LLC cells.
Treatment of LLC cells with low concentrations of bortezomib (100 ng/ml) inhibited NF-κ B activation and NF-κ B-dependent transcription, but not cellular proliferation. Bortezomib treatment of immunocompetent C57BL/6 mice bearing LLC-induced subcutaneous tumors and MPEs significantly blocked tumor-specific NF-κ B activation. However, bortezomib treatment did not impair subcutaneous LLC tumor growth, but was effective in limiting LLC-induced MPE. This specific effect was evidenced by significant reductions in effusion accumulation and the associated mortality and was observed with both preventive (beginning before MPE formation) and therapeutic (beginning after MPE establishment) bortezomib treatment. The favorable impact of bortezomib on MPE was associated with suppression of cardinal MPE-associated phenomena, such as inflammation, vascular hyperpermeability, and angiogenesis. In this regard, therapeutic bortezomib treatment had identical favorable results on MPE compared with preventive treatment, indicating that the drug specifically counteracts effusion formation.
These studies indicate that proteasome inhibition tailored to block NF-κ B activation of lung adenocarcinoma specifically targets the effusion-inducing phenotype of this tumor. Although the drug has limited activity against advanced solid lung cancer, it may prove beneficial for patients with MPE.
A malignant pleural effusion (MPE) affects one in seven patients with cancer, most commonly lung or breast adenocarcinoma [1, 2]. For these patients, MPE has severe consequences: it prohibits surgical cure and portends short and cumbersome survival . Current treatments against MPE (pleural evacuation or pleurodesis) [1, 2, 4, 5] are associated with hospital stay, interventional procedures, morbidity and mortality, and benefit only select patients [1–8]. Towards improving current practice against MPE, a better understanding of its pathogenesis is required, which may aid in developing effective strategies to block pleural fluid accumulation in patients with cancer.
The pathogenesis of MPE was unclear, mainly due to the lack of relevant animal models [9–11]. Using a prototype immune-competent mouse model, we described that activation of nuclear factor (NF)-κ B in lung adenocarcinoma specifically facilitates MPE, but not the growth or metastasis of this neoplasm [12, 13]. We found that this effect of NF-κ B on MPE formation was not mediated via enhanced tumor growth, but by enhanced expression of NF-κ B-dependent gene products, including tumor necrosis factor (TNF) and C-C motif chemokine ligand (CCL) 2 [14, 15]. We furthermore determined that this MPE-inducing phenotype of lung adenocarcinoma is not ubiquitous to all tumor types and involves specific MPE-associated phenomena such as inflammation, angiogenesis, and leakiness of pleural blood vessels [12–15]. These studies identified NF-κ B as a promising therapeutic target in lung adenocarcinoma-induced MPE.
Various approaches have been tailored to block NF-κ B in cancer cells, since the transcription factor has emerged as a promoter of inflammation-associated cancers [16–18]. These include blockade of inhibitor of NF-κ B (Iκ B) kinases (IKK)  and proteasome inhibition, which suppresses NF-κ B by diminishing Iκ B degradation . Although the latter approach is less specific, the proteasome inhibitor bortezomib blocks NF-κ B in a variety of cells and is already in clinical use against multiple myeloma [20–22]. Unfortunately, the initial optimism regarding activity of the drug against non small-cell lung cancer (NSCLC)  that led to several completed/ongoing phase I/II trials was recently hampered by the results of these trials reporting limited or no single-agent activity of bortezomib against advanced NSCLC [24, 25].
In the present studies we hypothesized that bortezomib treatment tailored to inhibit NF-κ B activation of Lewis lung cancer (LLC) is specifically effective in limiting MPE, but not solid tumor formation by this neoplasm. We tested our hypothesis by titrating the effects of bortezomib on LLC cell NF-κ B activation and proliferation and by conducting parallel experiments of intrapleural and subcutaneous introduction of this tumor into syngeneic mice.
Bortezomib (Millenium, Cambridge, MA) was purchased from the pharmacy, D-luciferin from Biosynth AG (Naperville, IL), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, passive lysis buffer, and firefly luciferase assay system from Promega (Madison, WI), recombinant human (rh) TNF from Peprotech (London, UK), ELISA from Peprotech (London, UK) and R&D Systems (Minneapolis, MN), anti-proliferating cell nuclear antigen (PCNA) antibody from SantaCruz Biotechnology (Santa Cruz, CA), terminal deoxynucleotidyl nick-end labeling (TUNEL) kit from Roche (Penzberg, Germany), anti-factor VIII-associated protein (F8A) antibody from Invitrogen (San Francisco, CA), and Evans' blue from Sigma (St Louis, MO).
LLC mouse lung adenocarcinoma cells were purchased from the American Type Culture Collection (Manassas, VA; identifier: CRL-1642), were verified by antigen H-2b expression, and were stored at -80°C, resuscitated, and cultured at 37°C in 5% CO2-95% air using DMEM 10% FCS supplemented with glutamine and 100 mg/l penicillin/streptomycin. Wild-type or pNGL LLC cells stably expressing a NF-κ B reporter (NF-κ B. GFP.LUC; pNGL) [12–14] were plated at equal densities in 6- or 96-well culture dishes till 20-30% confluent and were incubated with PBS or 1 nM rhTNF, in the presence of varying concentrations of bortezomib. Luciferase activity was determined after four hours by luciferase assay and bioluminescent imaging, as described below. Cell viability and mediator elaboration into culture media were determined after 24 hours, using MTS reduction capacity and ELISA, respectively. All cell experiments were done in triplicate.
Determination of luciferase bioactivity
Luciferase activity of whole cells or tissue homogenized in passive lysis buffer (Promega, Madison, WI) was determined using a "Junior" luminometer (EG&G Berthold, Bad Wildbad, Germany), after addition of 100 μl firefly luciferase assay system to 20 μl sample, as described previously . Luciferase activity was corrected for total protein, determined using the Bio-Rad assay (Hercules, CA). Serial bioluminescence imaging of live mice bearing pNGL LLC-induced subcutaneous tumors or MPE was done using intravenous injection of 1 mg D-luciferin, and of cultured pNGL LLC cells four hours after treatment application using addition of 10 mM D-luciferin [12, 13]. Imaging was performed using the Xenogen IVIS cooled CCD (Xenogen, Alameda, CA). Data were analyzed using Living Image v.2.50 (Xenogen) and IgorPro (Wavemetrics, Lake Oswego, OR) [12, 13].
Mouse TNF, C-X-C motif chemokine ligand (CXCL) 1, CXCL2, and CCL2 (detection limits: 5.1, 7.8, 1.5, and 15.6 pg/ml, respectively) expression levels were determined using ELISA and were corrected for total protein.
C57BL/6 mice were purchased from the Hellenic Pasteur Institute (Athens, Greece) and the Jackson Laboratory (Bar Harbor, ME) and inbred at the Animal Care facilities of the General Hospital Evangelismos (Athens, Greece) and of Vanderbilt University. Animal care and experiments were approved by the Veterinary Administration, Prefecture of Athens, Greece, and the Institutional Animal Care and Use Committee at Vanderbilt University and were conducted according to international standards http://grants.nih.gov/grants/olaw/GuideBook.pdf. Experimental mice were sex-, weight (19-24 g)-, and age (8-10 week)-matched. Solid adenocarcinomas and MPEs were generated, respectively, by injections of wild-type or pNGL LLC cells in the flank (5 × 105 cells) or pleural space (1.5 × 105 cells). In mice with subcutaneous LLC tumors, three vertical tumor dimensions (δ1, δ2, δ3) were measured weekly, and tumor volume (V) was determined using the formula V = π × (δ1 × δ2 × δ3)/6. In mice with LLC-induced MPEs, sacrifice, necropsy, and specimen (MPE, blood, tumor) collection were performed on day 14 as described previously [12–15, 26].
In vivo bortezomib treatment
Mice with subcutaneous LLC tumors received bi-weekly intraperitoneal bortezomib (100 ng/g = 0.1 mg/kg in 100 μl PBS) or 100 μl PBS control at days 2, 5, 9, 12, 16, 19, 23, and 26 after tumor cell inoculation. Mice that received intrapleural LLC cells were treated with intraperitoneal bortezomib (100 ng/g = 0.1 mg/kg in 100 μl PBS) or 100 μl PBS control starting either before (bortezomib prevention trial; days 2, 5, 9, and 12 after LLC cells), or after the onset of active fluid exudation (bortezomib regression trial; days 9 and 12 after LLC cells), which occurs at day 8 in the LLC-induced mouse MPE model [12, 26].
Histology & cytology
Tumors were fixed in 10% neutral buffered formalin (24 hours) and 70% ethanol (3 days). Tumors were dissected and embedded in paraffin. 5-μm-thick sections were cut, mounted on glass slides, and stained with hematoxylin & eosin. Alternatively, tissue sections were immunostained using antibodies for PCNA, TUNEL, and F8A, and immune-labeling was quantified, as described previously [12–15, 26]. For cytocentrifugal specimen (cytospin) preparation, 50000 pleural fluid cells were used. The slides were air dried, fixed in methanol for 10 seconds, and stained with May-Gruenwald-Giemsa. Distinct cell types were enumerated as a percentage of 400 cells on the slide.
Pleural Permeability Assay
At day 14 post-LLC cells, mice bearing MPEs induced by LLC cells received 200 μl of 4 mg/ml Evans' blue solution (0.8 mg) intravenously and were killed 1 hour later. Pleural fluid Evans' blue concentration was determined using absorbance at 605 nm, as described previously [12–15, 26].
All values are presented as mean ± standard error of mean (SEM). Survival is given as median (95% confidence interval) and was analyzed using Kaplan-Meier estimates. Differences in the means between two or multiple groups were examined using the Student's t-test or one-way ANOVA with least square difference post-hoc tests for normally distributed data, and the Mann-Whitney u-test or Kruskal-Walis test with Mann-Whitney u-post-hoc tests for not normally distributed data. Differences in survival were assessed by log-rank test. All P values are two-tailed; P values < 0.05 were considered significant. All statistical analyses were performed using the Statistical Package for the Social Sciences v.13.0.0 (SPSS, Chicago, IL).
Tailoring proteasome inhibition to block NF-κB activation of lung adenocarcinoma in vitro and in vivo
Specific suppression of lung adenocarcinoma-induced MPE formation but not tumor growth by NF-κB-targeting bortezomib treatment
NF-κB-tailored proteasome inhibition down-regulates proinflammatory gene expression of pleural tumors and cardinal MPE-associated phenomena
In the present studies we examined the effects of the proteasome inhibitor bortezomib on NF-κ B-dependent MPE induction by lung adenocarcinoma. By tailoring drug treatment to specifically target NF-κ B activation and not cell proliferation of this tumor we achieved consistent blockade of the transcription factor in cancer cells in vitro and in vivo. Delivered in this fashion, the proteasome inhibitor limited MPE formation by LLC cells without impacting their subcutaneous growth rate. In addition, therapeutic (eg, initiated before MPE formation) proteasome inhibition was equally effective in achieving MPE control and in prolonging survival with preventive treatment (eg, initiated after MPE establishment), suggesting specific anti-MPE effects of NF-κ B-targeted bortezomib treatment. Indeed, tailored bortezomib treatment suppressed NF-κ B-dependent gene expression of lung adenocarcinoma in vitro and in vivo, resulting in down-regulation of all major paracrine phenomena known to be involved in MPE formation: inflammation, vascular hyperpermeability, and neoangiogenesis.
This is the first preclinical study of the effects of any NF-κ B or proteasome inhibitor on MPE. Our results indicate that bortezomib is highly effective against experimental MPE, favorably impacting all outcome measures of MPE control. In addition, the drug was effective even when given after the onset of active lung adenocarcinoma-induced fluid exudation into the pleural space, implying the potential for therapeutic use against already established MPE. Human MPE is a significant clinical problem, a fact reflected by the overt insufficiency of current treatments  and by expert recommendations for upgrading its prognostic significance in the TNM staging system for NSCLC from a T4 to a M1a descriptor . In face of the above, our findings may prove clinically useful against human MPE.
In addition to their potential clinical utility, our results yield insights into the determinants of site-specific lung adenocarcinoma progression. In this regard, serosal involvement and dissemination of this tumor appears to be profoundly governed by a vicious triad of inflammation, vascular hyperpermeability, and new vessel formation, mechanisms that may be of lesser importance in solid tumor progression, where stimulus-independent tumor growth and evasion of apoptosis may be more pivotal . In this regard, the ability of tumor cells to induce a MPE differs between various tumor types  and may constitute an additional hallmark of adenocarcinoma. We show that this MPE-promoting phenotype of lung adenocarcinoma, heavily dependent on NF-κ B-controlled gene expression and not on NF-κ B-independent tumor growth [12–15], can be selectively targeted by tailored proteasome inhibition. The present and other lines of evidence suggest that targeting host-tumor interactions rather than tumor cell cycling may present an effective future therapeutic strategy against cancer . Compared with their activity against solid tumors, these approaches may be more effective against MPE, which is largely governed by the paracrine effects of tumor on the host immune system and vasculature [2, 9–15, 26]. Indeed, in our hands tailored proteasome inhibition specifically targeted lung adenocarcinoma-induced MPE formation but not the growth of this tumor in solid form. The specific anti-MPE effects of bortezomib were also evident by the fact that it was only effective when MPEs were present and preventive administration provided no additional benefit over therapeutic delivery. Although bortezomib enhanced apoptosis of pleural and subcutaneous tumors, this pro-apoptotic effect was of minor magnitude. In addition, bortezomib-induced apoptosis was observed in both subcutaneous and pleural tumors, and thus did not present a plausible explanation of the specific anti-MPE effects of the drug. However, bortezomib treatment limited paracrine phenomena specifically linked with MPE formation, such as angiogenesis, vascular hyperpermeability, and inflammation. Down-regulation of these mechanisms via suppression of NF-κ B-dependent genes in tumor cells may provide a more accurate mechanistic insight into the anti-MPE functions of proteasome inhibition.
Bortezomib has been tested against human NSCLC and has been found to be ineffective [24, 25]. However, our results suggest that the drug may be worth testing specifically against NSCLC-induced MPE. In this regard, trials against NSCLC included a majority of patients without MPE and a minority of patients with MPE [24, 25]. In addition, end-points related to MPE control were not included in the design of these studies. Hence a possible favorable effect of bortezomib treatment against MPE may have gone undetected and, despite the negative results of the aforementioned significant trials, bortezomib may be still worth testing against human MPE.
During the last few decades, knowledge of biologic mechanisms of disease has expanded tremendously. However, therapeutic targeting of single disease culprits is limited by biologic diversity and redundancy, as well as cost . In this regard, simultaneous suppression of multiple disease promoting pathways by small molecules would be advantageous . In our hands, a proteasome inhibition regimen designed to target NF-κB activation of lung adenocarcinoma limited MPE formation by suppressing the expression of multiple paracrine mediators of intrapleural adenocarcinoma dissemination and fluid extravasation, including TNF and CCL2 [14, 15]. Bortezomib treatment blocked the constitutive and inducible NF-κ B activation of mouse lung adenocarcinoma, leading to suppressed MPE-related inflammation, angiogenesis, and vascular leakiness, and hence providing for a microenvironment less permissive for MPE development [9–15].
Herein we showed the specific effects of NF-κB-tailored proteasome inhibition against MPE formation by mouse lung adenocarcinoma. In addition to previous genetic interventions, the pharmacologic approach employed herein strengthens the available data that support that serosal effusion formation, a hallmark of adenocarcinoma, is governed by expression of paracrine inflammatory and vasoactive mediators. Finally, we showed that tailored bortezomib treatment is highly effective against experimental MPE, the mouse analogue of a very common, difficult to treat, and debilitating occurrence in patients with cancer, setting a rational framework that supports the feasibility of clinical triage of proteasome inhibition against MPE.
The authors gratefully acknowledge financial support of this work by the "Thorax" Foundation, Athens, Greece (to IK and GTS) and the United States Department of Veterans Affairs (to TSB). The funding bodies had no involvement in study design, in the collection, analysis, and interpretation of data, in the writing of the manuscript, and in the decision to submit the manuscript for publication, which were in control of IP and GTS.
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