RETRACTED ARTICLE: Exemestane blocks mesothelioma growth through downregulation of cAMP, pCREB and CD44 implicating new treatment option in patients affected by this disease

Malignant Mesotheliomas (MM) are aggressive and lethal neoplasms arising from mesothelial cells lining the pleura, peritoneum, tunica vaginalis testis and pericardium. Human malignant pleural mesothelioma (MPM) is the most common form of mesothelioma that grows aggressively, with dissemination throughout the pleural cavity, and is frequently associated with massive pleural effusion [1]. MPM is considered to be closely associated with a personal history of prolonged exposure to asbestos fibres in patients [2, 3], although several etiologic factors iron [4] and simian virus 40 (SV40) [5] are reported to be involved in the development of MPM. The incidence of MPM is expected to increase at an alarming rate over the next few years, despite the banning of asbestos. Disease incidence varies markedly within and between countries. The highest annual rates of disease, approximately 30 case per million, are reported in Australia and Great Britain. The risk of disease increases with age and is higher in men. Time from asbestos exposure to disease diagnosis is on average greater than 40 years. Non occupational asbestos exposures contribute an increasing proportion of disease. With the exception of the United States, incidence continues to be on the increase. In developed countries peak incidence is expected to occur before 2030 [6, 7]. MPM, sometimes takes 10 years or more for changes to appear that are indicative of pleural disease, and even longer for symptoms to manifest. Patients frequently present respiratory symptoms, including dyspnea, shortness of breath and chest pain, extremely limiting the quality of life of the patients with this disease. Following diagnosis, most cases of malignant mesothelioma have poor survival [1]. The standard therapeutic modalities for MPM, including surgery, chemotherapy and radiation, have yielded unsatisfactory outcomes [8]. The combination of cisplatin and pemetrexed has become standard first-line therapy worldwide for patients who are not suitable for aggressive surgery, or in whom chemotherapy is recommended as part of a multimodality regimen with a mean survival of 12.1 months and 18.34 [9‐11]. Therefore, in order to improve the clinical outcome in the pharmacological treatment of this refractory tumour, drugs aimed at targeting novel and/or characterized tumour-specific cellular targets are needed. The pathogenic mechanisms underlying mesothelioma involve epigenetic gene regulation [12] and deregulation of multiple signaling pathways, including sonic hedgehog signalling [13, 14], activation of multiple receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) family and MET, and subsequent deregulations of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)-AKT signaling cascades, the TNF-α/NF-κB survival pathway, Wnt signaling, and loss of tumor suppressors such as Neurofibromatosis type 2, p16INK4A, and p14ARF [15, 16]. Understanding the mechanisms of the dysregulated signaling pathways allows strategies for development of targeted new therapies against this devastating disease.

Recently, we have demonstrated the presence of aromatase (CYP19A1) in MPM cell lines and tumor tissue sections from patients with MPM [17, 18]. CYP19A1 was expressed in the majority of MPM samples as a cytoplasmic protein and the cytoplasmic expression of CYP19A1 significantly correlated with poor survival [17]. The World Health Organization classifies MM into epithelial, sarcomatoid, and biphasic types, each of which can be subdivided further. This classification has implications for both diagnosis and prognosis [19]. Prognosis is poor for all MMs, but sarcomatoid MMs have a particularly poor response rate to treatment: a significant association between high expression of CYP19A1 and sarcomatoid MPMs was found [17]. These observations suggested that CYP19A1 plays a role in tumour progression in MPM. CYP19A1 is a key enzyme in the biosynthesis of estrogen (converting testosterone into estradiol (E2). Females were identified as being a positive prognostic factor for peritoneal MM: female patients have a median survival than males (17.3 months compared to 11.8 months respectively) [20, 21]. The estrogen receptors expression using immunohistochemistry, was demonstrated in peritoneal tumors and not in pleural tumors [22]. Recently, immunohistochemical analysis revealed intense nuclear ERβ staining in normal pleura that was reduced in MM tissues. Conversely, neither MM nor normal pleura stained positive for ERα [23]. This leads to more carefully explore the role of estrogen in the pathogenesis of MM and especially on MPM.

Exemestane an inhibitor of CYP19A1 type 1 (steroidal inactivator), induces cell death in Ist Mes1, Ist Mes2 and MPP89 MPM cell lines [17]. This initial finding has provided the impetus for the studies presented here, aimed to investigate the mechanism of action of exemestane on MPM cells and xenograft MPM models. We have thus identified possible pathways between cyclic adenosine monophosphate (cAMP), cAMP response element-binding protein (CREB), CD44 and pAKT, Bcl-xL that are downregulated by exemestane in MPM cells sensitive. Finally, we demonstrated that exemestane was effective in vivo alone and in combination with pemetrexed, and that the effect of this association was superior compared to the therapeutic combination cisplatin/pemetrexed.

The current study highlights the effect of exemestane and its potential translation into the clinical setting for the treatment of MPM. A recent study reported the presence of CYP19A1 in cells and tissues of MPM and the antiproliferative action of exemestane in Ist-Mes1, Ist-Mes2 and MPP89 [17]. Normal mesothelium exhibited a weak positivity for CYP19A1 and the human pleuralmesothelial cell Met-5A does not express appreciable levels of CYP19A1 by western blot. Met-5A was not sensitive to exemestame treatment. In order to better understand the mechanism of action of exemestane in vitro and in vivo we studied other two MPM cell lines, MSTO tumorigenic in mice and NCI. Exemestane 35 μM was found to inhibit the growth of MSTO cells in vitro, inducing arrested cell-cycle progression abrogated the tumor cell migration and reduced the colony formation capacity (Figure 1). On the contrary, nothing like what was observed in NCI, therefore this line was defined cell resistant to 35 μM of drug. In vitro experiments performed on MPM sensitive cell lines (MSTO, Ist-Mes1, Ist-Mes2 and MPP89) and NCI resistent cell lines upon exemestane treatment have helped us to identify drug targets. The exemestane dosage for all experiments was of 35 μM. Although the selected concentrations seem to be high, similar concentrations of an aromatase inhibitor have also been used in previous studies for in vitro experiments [24, 26]. The dose of exemestane currently used in clinical practice is 25 mg daily. Exemestane exhibits an excellent safety profile in humans, having no significant toxicity at doses up to 600 mg/day and it is exceptionally well tolerated [27]. At clinically administered doses, the plasma half-lives of exemestane was 27 hours [28]. Exemestane is an irreversible, steroidal aromatase inactivator, structurally related to the natural substrate androstenedione. It acts as a false substrate for the aromatase enzyme and is processed to an intermediate that binds irreversibly to the active site of the enzyme causing its inactivation, an effect also known as “suicide inhibition” [29].

Another possible mechanism includes changes in aromatase activity through a cAMP-dependent mechanism [30]. A previous study reported an increase of cAMP levels in lung cancer cell lines, 15 min after treatment of cells with exemestane. This effect was reversed 30 min after the application of exemestane [24]. MPM cell lines sensitive upon 30 min exemestane treatment exhibited decreased levels of cAMP levels. This difference could be due to the different tissue types of origin. cAMP is a ubiquitous second messenger. Many growth factors and hormones regulate cellular activity through second messengers which correspondingly induce multifunctional protein kinases [31].

Activation of cAMP signaling involves binding of an extracellular ligand to a GPCR which through G proteins regulates one of several isoforms of adenylyl cyclase (AC) leading to cAMP generation. Although other effectors of cAMP have been identified, the most common downstream effector system is cAMP-dependent protein kinase (PKA). In its inactive state, the PKA holoenzyme consists of two catalytic (C) subunits bound noncovalently to a regulatory (R) subunit dimmer [32]. Binding of four cAMP molecules, two to each R subunit, leads to a conformational change and dissociation into an R subunit dimer with four cAMP molecules bound and two C monomers [33]. The C subunits then become catalytically active and phosphorylate serine and threonine residues on specific substrate proteins [34]. When cAMP rises, the C subunit released from the holoenzyme enters the nucleus by passive diffusion where it regulates a number of cellular processes, including motility, metabolism, neurotransmitter release, and transcription by the reversible phosphorylation of key substrates [35]. cAMP regulates the expression of specific genes by mediating the PKA-dependent phosphorylation of the CREB transcription factor in a 30-min period [31]. In MPM cell lines treated with exemestane, we found a direct correlation between levels of cAMP and expression of p-CREB. In particular the lines sensitive to exemestane treatment showed levels of cAMP and p-CREB decreased compared to controls untreated while in the line NCI resistant cAMP levels and p-CREB increased also in the presence of the drug (Figure 2A, B).

Exemestane inhibits cell migration in MPM cell lines sensitive to the agent. Data report CD44 responsible for cell migration in MM [25, 36]. CD44 is a type I transmembrane glycoprotein (85–200 kDa) and functions as the major cellular adhesion molecule for hyaluronic acid, a component of the extracellular matrix. CD44 is expressed in most human cell types and is implicated in a wide variety of physiological and pathological processes, including lymphocyte homing and activation, cell migration, tumor cell growth, metastasis [37] and chemoresistance [38]. Flow cytometry (Figure 2C) revealed that MPM cell lines highly expressed CD44 and exemestane treatment reduces its levels in all lines except in NCI. The silencing of CD44 in MSTO and NCI confirms the importance of CD44 in cell migration and suggests its essential in the response to the drug, although the direct target of exemestane might be a factor upstream of CD44 that in NCI gives it resistance. Given the involvement of pAKT, Bcl-xL in Ist-Mes1, Ist-Mes2 and MPP89 drug response [17], we decided to assay their expression and p-CREB in CD44 silenced MSTO and NCI cells upon exemestane treatment and control. As shown in Figures 2E, p-CREB, pAKT and Bcl-xL expression were reduced by treatment with exemestane only in MSTO (silencied or not) indicating once again that the phosphorilation of these could be a target of the drug. Altogether, these data suggest that exemestane, reducing the levels of estradiol, affects cAMP and phosphoinositide 3-kinases (PI3K) pathway (Figure 4). Estrogen acts via the regulation of transcriptional processes, involving nuclear translocation and binding on specific response elements, thus leading to regulation of target gene expression. However, the observation of the effects induced by steroid hormones that are too fast to be mediated by the activation of RNA and proteins, has led to the identification of non- transcriptional mechanisms of signal transduction through steroid hormone receptors. These so-called “non-genomic” effects involve steroid-induced modulation of cytoplasmic or of cell membrane-bound regulatory proteins. Relevant biological actions of steroids have been associated with this signaling in different tissues. Ubiquitary regulatory cascades such as MAPK [39], the phosphatidylinositol 3-OH kinase (PI3K) and tyrosine kinases [40] are modulated through non-transcriptional mechanisms by steroid hormones. Steroid hormone modulation of cell membrane-associated molecules such as ion channels and G-protein-coupled receptors (GPCR) has been shown in diverse tissues [41]. Lines of evidence suggest that the estrogen-mediated activation of AC occurs independently of known ERs but rather requires GPR30 protein [42]. Since the exemestane induces a modulation of cAMP in short time (30 min) and considering that cAMP is produced upon AC activation, we ignored the classical estrogen receptors and focused on GPR30.

The GPR30 receptor binds estrogen and cause estrogen-mediated AC stimulation and PI3K cascade. PI3K are family of lipid kinases capable of phosphorylating the 3’OH of the inositol ring of phosphoinositides. They are responsible for coordinating a diverse range of cell functions including proliferation and cell survival. PI3K phosphorylates AKT (pAkt) which in turn regulates Bcl-xL, anti-apoptotic protein. The AC catalyzes the formation of cAMP which stimulates PKA which in turn activates CREB (pCREB). pCREB translocates into the nucleus and acts as a transcription factor for several genes including CD44. PI3K and cAMP pathways inhibition by exemestane causes pAKT, BclxL, pCREB and CD44 down-regulation in MPM cell resulting in cell death. Intuitively, it can be hypothesized that the action of exemestane is achieved by reducing the levels of estradiol, its binding with GPR30 and related downstream pathway (Figure 4). Further studies are underway to validate the role of GPR30 in MPM. Using a MPM xenograft animal model resulting from the subcutaneous injection of MSTO cells, we compared the effect of exemestane on tumor growth in immunodeficient mice versus to control. Therapy was initiated after tumor establishment. Mice in both groups were followed for tumor size and toxicity. Treatment with exemestane induced tumor growth delay (Figure 3A) and decreased tumor mass completely in 70% of males and 80% of females as compared with control. Altogether, the impact of exemestane on MPM xenografts mirrors the effects noted in cell culture. Previous work shows the effectiveness of pemetrexed and cisplatin in MPM cell lines. Studies with the MSTO-211H cell line showed synergistic effects when pemetrexed was combined concurrently with cisplatin [43‐45]. Pemetrexed is the first and only chemotherapy agent that has been granted marketing approval for use in combination with cisplatin for the treatment of chemonaive patients with unresectable MPM. Although pemetrexed combined with cisplatin showed a significant survival prolongation compared with cisplatin alone, the difference was only 2.8 months [46]. It is therefore necessary to augment the therapeutic effect of pemetrexed to further improve the survival of MPM patients. Therefore, given the growing body of clinical evidence suggesting only minimal effects of monotherapy, we decided to test the association exemestane-pemetrexed compared to standard combination cisplatin-pemetrexed in nude mice. Although the two treatments are effective in reducing the tumor mass, the association exemestane-pemetrexed was significantly (P = 0.039) more effective than cisplatin-pemetrexed (Figure 3B). In the present study, we clearly showed the increased efficacy of exemestane alone or in combination with pemetrexed versus the standard therapy pemetrexed-cisplatinum against MPM in the xenograft implantation model. In this context should not be underestimated that exemestane was as effective as the combination pemetrexed-exemestane, therefore exemestane monotherapy could be very beneficial to MPM patients.

We could not do a histologic examination to clarify the mechanism of the increased efficacy of exemestane and conventional chemotherapy because the therapy was so effective that we could not obtain enough tumor samples. These findings are encouraging and possibly support further investigation of exemestane in the clinical MPM context and highlight the opportunity to test, in the experimental MPM model, new compounds active with the same mechanism of action [47]. Exemestane is widely used in the treatment of breast cancer. It is active clinically in preventing, delaying progression of, and treating mammary cancers, many of which are estrogen receptor-positive. Exemestane 25 mg orally once daily was generally well tolerated without major toxicity [48] and it displays anti-inflammatory properties [49]. Given that exemestane has already been approved, it may proceed rapidly in clinical trials. After evaluating the benefits of exemestane alone or in association with chemotherapy in a group of patients, this therapy can be applied in MPM treatment protocols. Exemestane could possibly open new treatment strategies in association with standard therapy for patients afflicted with MPM. At present there are no clinical trials on the inhibitors of CYP19A1 in MPM, this may be probably due to the recent identification of CYP19A1 in MPM. In lung cancer, where studies of CYP19A1 are at a more advanced stage, some clinical studies consider the inhibitors of CYP19A1 an anti-oestrogen.