Malignant Mesothelioma: Development to Therapy

Abstract

Malignant mesothelioma (MM) is an aggressive cancer of the mesothelium caused by asbestos. Asbestos use has been reduced but not completely stopped. In addition, natural or man-made disasters will continue to dislodge asbestos from old buildings into the atmosphere and as long as respirable asbestos is available, MM will continue to be a threat. Due to the long latency period of MM development, it would still take decades to eradicate this disease if asbestos was completely removed from our lives today. Therefore, there is a need for researchers and clinicians to work together to understand this deadly disease and find a solution for early diagnosis and treatment. This article focuses on developmental mechanisms as well as current therapies available for MM.

Introduction

Malignant mesothelioma (MM) is a cancer of the mesothelial lining of the body caused by asbestos. In the US, asbestos was used for various purposes including building materials until the 1970s, suggesting that old buildings still have asbestos. Asbestos fibers are friable when damaged, leading to smaller fibers which can become airborne when disturbed. This is of particular interest because asbestos is only harmful when airborne fibers are inhaled. Natural disasters like tornados and man-made disasters such as the terrorist attacks on September 11, 2001 can rip apart old buildings resulting in airborne asbestos. There are various types of asbestos fibers, all of which are proven carcinogens in human beings. Respirable asbestos fibers, once inhaled, can cause serious damage to the lung, resulting in lung fibrosis (asbestosis) or MM. Asbestos fibers have also been shown to reach the pleural cavity and directly injure mesothelial cells. The injury to mesothelial cells may result in activation of inflammatory pathways including inflammasomes and the release of inflammatory cytokines. By autocrine and/or paracrine pathways, these cytokines and other signaling molecules can cause mesothelial to fibroblastic transformation (MFT) and finally transformed mesothelial cells can give rise to MM (Figure 1).

Figure 1.

A schema showing development of malignant mesothelioma (MM) in response to asbestos exposure. Inhaled asbestos fibers first encounter tracheal and lung epithelial cells as well as alveolar macrophages which attempt to clear the fibers. Reactive oxygen species (ROS), inflammatory mediators as well as signaling pathways that are activated in response to the fibers may be involved in sending signals to the pleura to initiate transformation of pleural mesothelial cells. Asbestos fibers may also be carried into the pleural cavity by direct transfer or by the lymphatic system where they may come into direct contact with the mesothelial cells. Chronic inflammation and growth factor signaling in combination with factors thus released by the mesothelial cells and pleural macrophages may lead to the initiation of transformation (mesothelial to fibroblast transition (MFT)) events that eventually lead to tumorigenesis and development of malignant mesothelioma.

In the present prospect we discuss in detail how asbestos fibers can reach the mesothelial cells and cause their transformation, resulting in MM development. In the latter part of this review, we touch upon proposed and current biomarkers and therapies for the diagnosis and treatment of MM.

Asbestos

Asbestos is the commercial name for a group of six naturally occurring silicate mineral fibers (actinolite, anthophyllite, chrysotile, cummingtonite-grunerite (amosite), crocidolite and tremolite) that each demonstrate longitudinal parting into very thin fibrils. Prior to the 1970’s, asbestos was a globally popular construction material due to its resistance to heat, fire, electricity and chemical damage. Unfortunately, with the increase of asbestos use came an increase in asbestos related diseases such as asbestosis and MM. Crocidolite (also called blue asbestos) is regarded as the most carcinogenic of asbestos types in part because of its durable nature and rod-like shape [1]. Once lodged in the lungs, asbestos fibers move to locations such as the pleura by unconfirmed mechanisms and cannot be naturally expelled from the body. It is theorized that the asbestos could be redistributed to the body cavities in two ways: by fibers physically moving to the outside of the lung tissue and being picked up by the pleura and/or through fibers being picked up by the lymphatic and/or blood systems in an attempt to clear the foreign material [2].

The molecular pathogenesis of MM is still an elusive multifactorial event involving multiple mechanisms. Many aspects of asbestos fibers; such as the length and shape of the fiber, durability, chemical composition and biopersistance promote disease and carcinogenesis [3]. Both local and more distant responses are involved in the disease progression of mesothelioma. Local to asbestos deposits, phagocytic cells attempt to phagocytose the asbestos fibers, which leads to frustrated phagocytosis due to the high aspect ratio (length to diameter ratio) of the fibers. In conjunction with this process of frustrated phagocytosis, reactive oxygen species (ROS) are generated leading to increased growth factor signaling, proliferation and signal transduction pathway stimulation in response to local tissue damage [4]. One can imagine how this multifaceted environment of tissue damage, growth and survival creates an environment that promotes cell transformation.

Mesothelial Cells

Asbestos exposure and injury to mesothelial cells results in MM development. The mesothelial cell, a specialized type of cell that makes up the protective layer of tissue called the mesothelium, is mostly flat and thin [4]. The primary function of the mesothelial cell is to form a protective monolayer over the internal organs, thereby providing a non-adhesive surface supporting organ movement aided by production of a lubricating fluid. Mesothelial cells are functionally diverse cells with multiple functions and properties. The importance of these cells in reference to normal organ function is supported by the fact that injury to the mesothelium can cause organs to adhere to the serosal wall consequently leading to the restriction of movement within the affected cavity [5]. Examples of this would be the restriction of lung movement, breathing capabilities and occasionally, cardiac function caused by injury to the pleural mesothelium, such as is seen in late stage MM.

In addition to the barrier functions of the mesothelial cell, these cells are also involved in the transportation of fluid and cells across serosal cavities, antigen presentation, immune surveillance, cytokine and chemokine production, inflammation, wound healing, coagulation, fibrinolysis and tumor cell adhesion [5, 6]. Once believed to only provide a barrier to protect the inner organs; the mesothelial cell has proved to be a cell with an astounding number of capabilities that could provide information on more advanced treatment for mesothelial diseases such as MM. Mesothelial cells are capable of initiating cell proliferation, wound repair, differentiation, migration, and inflammation via the release of molecular mediators. Some of these mediators include cytokines, chemokines, growth factors and matrix components [5].

The mesothelial cells secrete a lubricant consisting of glycosaminoglycans and phosphatidylcholine to facilitate smooth organ movement and further protect against invading organisms and abrasive damage [5]. The lubrication secreted by mesothelial cells has also been linked to cancer prevention. To this effect, hyaluronan, a glycosaminoglycan secreted by mesothelial cells, has been shown to prevent ovarian tumor cell attachment to peritoneal mesothelial cells [7].

Effect of asbestos exposure on mesothelial cells

The result of asbestos exposure of the lung in inhalation studies has shown that asbestos induces an acute inflammatory response locally around fibers. This acute inflammatory response includes the release of pro-inflammatory cytokines, macrophage and neutrophil recruitment, airway epithelial cell proliferation, and later mesothelial cell proliferation [8]. As discussed previously, asbestos is capable of moving from the lung to the pleura to affect the mesothelial cells that lay there. It has been shown that long asbestos fibers are more durable than shorter fibers and can cause chronic inflammation and repeated injury to pleural mesothelial cells [9]. It is believed that this chronic inflammation from asbestos exposure would predispose local mesothelial cells to carcinogenesis. Recent supporting data from Xu et al (2012) demonstrated the presence of crocidolite asbestos fibers, administered by intrapulmonary spraying, in macrophages of the pleural cavity lavage fluid [10]. In this study crocidolite asbestos exposure induced hyperplastic proliferative lesions of the visceral mesothelium as well as abundant inflammatory cell infiltration. This suggests that inflammatory reactions in the lung and pleural cavity were responsible for the proliferative lesions seen in the pleural mesothelium.

Although basic mechanisms of how asbestos exposure leads to mesothelial cell proliferation/transformation and development of MM is not clear, several in vitro studies by our group and others have shed light on the possible mechanisms involved in the process. Using microarray, gene or protein pathway arrays, we and others have reported a special signature of gene expression in mesothelial cells exposed to asbestos [11]. Wang et al. (2011) found about 21 proteins/phosphoproteins that were dysregulated, mostly associated with EGFR/ERK and PI3K/AKT pathways [12]. Our group on the other hand, used microarray technology and human mesothelial cells exposed to either crocidolite asbestos or Libby six-mix. Libby six-mix caused alteration in several genes, the most prominent being Superoxide dismutase (SOD). In addition to up-regulating SOD, Libby six-mix also caused increased production of oxidants and a transient decrease in reduced glutathione (GSH) [13], suggesting that Libby six-mix affects human mesothelial cells by altering their oxidative environment. Yet another study from our group demonstrated that crocidolite asbestos exposure can cause an altered profile of genes in human peritoneal mesothelial cells. With crocidolite asbestos Activating Transcription Factor (ATF3) 3, a cyclic AMP response element binding protein (CREB) family member was the highest expressing gene [14]. Furthermore, down regulation of ATF3 by siRNA (small interfering RNA) caused a significant decrease in secreted levels of pro-inflammatory cytokines (IL-1β, IL-13, G-CSF) and growth factors (VEGF and PDGF-BB). Findings from this study again emphasize the role of asbestos-induced inflammation in mesothelial cell injury and subsequent carcinogenesis. Another member of this family, CREB1 was also activated by asbestos in human mesothelial cells, and human MM cells and tumors showed constitutive activation of CREB [15]. Detailed work using siRNA to silence CREB1 revealed its role as a pro-survival protein acting via Bcl2 up-regulation. Follow up in vivo studies from our group confirmed that CREB1 regulates MM tumor growth predominantly by regulating inflammation (unpublished data).

Another signaling pathway that is studied extensively by our group is extracellular signal regulated kinases (ERKs). ERKs are modulated by asbestos in mesothelial cells and may be responsible for causing MMs. Crocidolite asbestos exposure of telomerase immortalized human mesothelial cells (LP9) and SV40 transformed human mesothelial cells (MET5A) caused activation of ERK1/2 via epidermal growth factor receptor (EGFR). Furthermore, silencing of ERK1, 2 or AKT by siRNA demonstrated that asbestos-induced cell death is ERK1/2 dependent in both cell lines [16]. It is also noted in this study that MET5A cells were more resistant to asbestos-induced toxicity than LP9 cells. The increased resistance to asbestos in MET5A cells can be attributed to elevated levels of calretinin, as elevated calretinin levels strongly correlate to enhanced asbestos resistance [17]. Recently we have shown another ERK, ERK5 to be activated by asbestos in human mesothelial cells and may play a role in the development of MM [18].

While altering molecular expression and activation in mesothelial cells, asbestos also produces a significant amount of cell death. Mesothelial cell death by asbestos has been shown to contain a regulated form of necrosis that causes the release of high-mobility group box 1 (HMGB1), (an inflammatory protein usually located in the nucleus) into the extracellular space. Mesothelial cells as well as macrophages secrete tumor necrosis factor alpha (TNF-α) in response to HMGB1-induced inflammation, activating NF-κB [19]. NF-κB is part of a survival pathway that allows some of the mesothelial cells exposed to asbestos to survive and potentially transform into MM cells [20]. Our unpublished data further show that asbestos-induced HMGB1 secretion from human mesothelial cells is NLRP3 (NOD like receptor protein 3) inflammasome dependent. This study is also first to demonstrate that asbestos can prime and activate NLRP3 inflammasomes in mesothelial cells, resulting in IL-1β and IL-18 release which may be responsible for transforming mesothelial cells in an autocrine manner. With this short review of current pathways involved in mesothelial cell exposure to asbestos it becomes clear that the mesothelial cell is diverse and complicated in its capacity to react to harm. Understanding the mechanisms of transformation of mesothelial cells by asbestos may provide enlightenment of the possible pathways responsible for development of MM and should be considered as potential targets.

Mesothelioma

MM is an asbestos-associated malignancy of mesothelial cell that is typically diagnosed at a late stage with a poor prognosis (median survival: 9–13 months) [21]. Work related asbestos exposure is the major cause of MM [22]. Most commonly, MM arises in the pleural area of the mesothelium surrounding the lungs, but it can also infrequently develop in the mesothelium of the peritoneum, pericardium and tunica vaginalis. After initial exposure to asbestos, MM development can take 20–60 years to manifest. The evidence that asbestos is a direct and major cause of MM is overwhelming starting with Wagner et al in 1960 [23] and continuing today with countless studies containing supporting evidence.

It remains unclear why asbestos exposure leads to MM in certain individuals, while others do not develop the devastating disease. Although 70–80% of MMs are caused by work-related exposure to asbestos, only about 5% of those exposed to asbestos develop MM [24]. These observations are indicative of an individual characteristic pre-disposing certain populations to become susceptible to MM development when exposed to asbestos. Genetic factors can play a role in the development and progression of MM as depicted in a study on the population in parts of Cappadocia, Turkey exposed to erionite (a fiber found in the volcanic rock of this area that shares many similar properties with crocidolite asbestos). Erionite related MM reached epidemic proportions, but only in families that passed on a predisposition to MM by erionite exposure in an autosomal-dominant manner [25]. More recent studies have shown that a significant number of sporadic MM cases exhibit somatic BRCA 1 associated protein (BAP1) mutations while cases of familial MM were found to have germline BAP1 mutations [26, 27]. In the case of the individuals with familial MM, BAP1 germline mutations appeared to predispose them to develop MM after exposure to very low levels of asbestos found in construction materials in their homes [26]. Together, these studies indicate that genetic factors such as BAP1 mutations contribute to a predisposition to develop MM after asbestos exposure.

The annual incidence rate of MM in the United States is approximately 3300 cases per year [28]. In the UK the continually rising peak is projected to begin its descent in 2015. Japan, on the other hand, is not projected to peak until the year 2027; according to a recent study looking at occupational exposure to asbestos [29]. Numbers of MM diagnoses in Japan began to climb in the year 2000 due to late asbestos use restriction in addition to a lack of proper ventilation and protective equipment to protect from asbestos exposure. Like the US, Japan has also not banned the use of asbestos entirely, only restricted its use, thus not eliminating future risk. Large areas of the world remain that have not restricted the use of asbestos and fail to provide appropriate protective equipment.

Asbestos use has by and large caused this worldwide problem of asbestos-related diseases that continues today. The WHO estimates that 125 million people worldwide are currently exposed to asbestos in the workplace [30]. Even in locations where asbestos is banned, people continue to be at risk for asbestos exposure. Older buildings that still contain asbestos are a potential hazard to local populations and rescue workers. History shows us that, in cases of natural disasters or explosions, asbestos from buildings can become airborne, contaminating the air with asbestos fibers that can be inhaled by individuals in the surrounding area. One such example is the fall of the twin towers in the United States. Rescue workers and the local population were exposed to asbestos from the air as well as fibers that had settled to the ground from damaged construction material. The asbestos-exposed population is now at risk for developing MM and other asbestos-related diseases. Factors that predispose specific individuals but not others to MM require further study to be elucidated. Such studies may help provide new chemotherapeutic targets to improve the treatment of MM and increase the median survival after diagnosis.

Current MM treatment options

Malignant mesothelioma (MM) is refractory to most treatment options currently available and its diagnosis also poses a great challenge to pathologists [31]. Current treatment options include surgical resection of the tumor (e.g. radical pleurectomy (RP) or extrapleural pneumonectomy (EPP)) if operable, chemotherapy and radiotherapy as single agents or as part of a multimodal approach [4]. These options do not improve survival substantially. Additionally, small molecule inhibitors of growth factor signaling pathways such as epidermal growth factor receptor (EGFR) and mitogen activated protein kinase kinase (MEK) among others, are also being investigated for use as chemotherapeutics for MM. Targeted delivery of chemotherapeutics as well as immunotherapeutics are also in development for the management of MM [4].

MM is often diagnosed in the late stages when the disease is too advanced for current therapies. This unfortunately leaves palliative care as the only option. While EPP facilitates complete resection of all diseased tissue from the pleural cavity, the number of MM patients that qualify to undergo this radical surgery is limited by their cardiopulmonary function test outcomes [32]. In EPP, resection of the affected lung en bloc with the pericardium and parietal pleural tissue is conducted to achieve complete removal of visible tumor mass [33]. However, because it is difficult to assess whether all malignant cells have been removed and MM has a history of local reoccurrence even after surgery, EPP and RP are now combined with chemotherapy or radiotherapy to improve the survival rate and extend time to recurrence [32, 33]. Chemotherapeutics that are approved for treatment of MM include permetrexed and cisplatin [34, 35]. On the other hand, protease inhibitors like bortezomib and monoclonal antibodies against vascular endothelial growth factor (VEGF) are in phase II trials as part of combination therapies with cisplatin and permetrexed [36, 37]. Unfortunately, VEGF inhibitors have failed to improve survival [36] while therapies including bortezomib may have some promise [37]. The inability of the above mentioned treatment options to improve survival after diagnosis with MM beyond 12 months combined with the lack of biomarkers that enable the early detection of MM necessitates the urgent need for the discovery of more effective treatment modalities and biomarkers.

Preclinical studies and trials are being conducted to develop better management options for MM. These include the study of antigens over-expressed in MM and other cancers, like mesothelin and podoplanin. Over-expression of podoplanin and mesothelin has been shown to be important for adhesion and viability of MM cell lines [38, 39]. Mesothelin (MSLN) is a differentiation antigen that is expressed on the surface of normal mesothelial cells and over-expressed in MM, pancreatic and ovarian cancer. MSLN has been exploited as a means of targeting MM and other MSLN over-expressing cancers through the conjugation of the MSLN antibody to an immunotoxin [40, 41], as well as for targeted drug delivery and immunotherapy. Recent work from our group has demonstrated a targeted drug delivery method for the delivery of doxorubicin (Dox) using acid prepared mesoporous silica particles (APMS) loaded with Dox and functionalized with mesothelin antibodies that targeted to tumor cells in in vivo studies thereby reducing the toxicity of doxorubicin [42]. Mesothelin itself has a pro-proliferative effect on MMs and knocking it down with siMSLN decreases the viability of the MM cell lines [43].

An additional antigen being investigated as a target for the development of MM immunotherapy is podoplanin. Podoplanin was first discovered in podocytes and has been shown to be over-expressed in some MM cell lines with increased invasive phenotypes [44]. By treating MM cell lines with high expression of podoplanin with the human–rat antibody chimera, NZ-8, Abe et al [39] were able to demonstrate increased antibody dependent cellular cytotoxicity (ADCC) as well as increased complement dependent cytotoxicity in vitro specific to cells over-expressing podoplanin only. [39]. The ADCC observed in the MM cell lines was also shown to be mediated by human mononuclear cells as opposed to rat NK cells in the case of the rat antibody NZ-1 in vivo using subcutaneous SCID mice models of MM [39].

While antibody mediated therapies are being developed and may prove effective with low side effects, such therapies will only be effective for MM patients whose tumors over-express the requisite antigens. As such, other targets that are more common to the majority of MM tumors are needed. Targeting signaling pathways essential to the survival/proliferation of MM tumors would also help improve treatment outcomes, especially when used as part of a multi-treatment modality [45]. Small molecule inhibitors against EGFR, PI3K or MEK have been shown to reduce tumor size in preclinical studies when used together [46]. Combination treatment therapies will help combat the refractory nature of MM tumors and delay the development of resistance since multiple targets are being utilized [47]. Our recent work has demonstrated the significant role of ERK5 in MM tumorigenesis and projected it as a potential therapeutic target [18]. Furthermore, knockdown and inhibition of ERK 1 and 2 has also been demonstrated to reduce tumor growth rates in mouse tumor xenograft studies while increasing the sensitivity of different MM cell lines to Dox [48]. The transcription factor, CREB1, has also been found to be constitutively activated in human MM cells and tumors where it conferred protection from apoptotic cell death as evidenced by an increase in Dox-induced cell death after silencing of CREB1 with siCREB [15]. Therefore, identifying and employing inhibitors of CREB as part of a multimodal therapeutic approach to the treatment of MM could help improve survival outcomes. The receptor tyrosine kinase Eph receptor B4 (EphB4), important for a variety of developmental processes and over-expressed in MM [49], has recently been shown to be a potential therapeutic target for treating MM [50]. In that study, Liu et al. showed that the EphB4 inhibitor, sEphB4-HAS, used alone or in combination with the VEGF inhibitor, Bevacizumab, was effective at reducing tumor volume, angiogenesis and proliferation of cells in subcutaneous mouse xenograft models of human sarcomatoid MM (H2373 MM cells) [50].

Another way of improving the early diagnosis of MM would be the discovery of a panel of biomarkers. Validated biomarkers would enable the early detection of MM in patients with the hope that early detection would facilitate complete resection of small tumors and more effective treatment of responsive early stage MM tumors. The few biomarkers available are still being validated and are yet to be proven as concrete prognostic and detection tools for MM. Mesothelin, the most studied MM biomarker to date, is found circulating in the serum of MM patients and recent studies indicate that the soluble mesothelin related peptide (SMRP) detected in pleural effusions is effective at distinguishing MM from other pleural effusion causes and holds promise as a marker for monitoring disease progress/treatment efficacy [51, 52]. One other gene product of the MSLN gene, megakaryocyte potentiating factor (also known as N-ERC) has been shown to be equally as effective as MSLN/SMRP at distinguishing MM pleural effusions from non-malignant and other pleural effusions [53]. Although MSLN and the other mesothelin related proteins are highly specific for MM, they are not over-expressed by the poorly differentiated sarcomatoid MM subtype [53]. This limits their use as biomarkers for all MM subtypes. To this end, other biomarkers that are widely expressed in all or most MM subtypes are under investigation.

Osteopontin (OPN), a widely expressed cancer antigen, has been studied as a potential diagnostic tool for the early detection of MM. OPN is a cell surface sialoprotein that is involved in bone matrix formation and tumor invasion among other functions [reviewed in 54]. OPN is over-expressed in a number of cancers including MM, lung and breast cancer [55, 56]. Due to the presence of a thrombin cleavage site on OPN however, variable results have been obtained when testing serum from MM patients [reviewed in 57]. Moreover, HMGB1, an inflammatory protein that also plays a role in transcription, proliferation and DNA repair is found to be elevated in the sera of MM patients and has been shown to be of great diagnostic value for both malignant pleural and peritoneal mesotheliomas [58]. As HMGB1 is considered to be a general marker of tissue injury and inflammation, its specificity for MM needs to be validated. Recently, Pass et al showed that Fibulin-3, a basement membrane protein of mesenchymal origin [59], is a specific biomarker of MM in blood and pleural effusion samples from MM patients and matched controls. A blinded validation study also confirmed the specificity of this biomarker [60]. However, since the discovery of Fibulin-3 as a biomarker for the detection MM is fairly recent, more rigorous validation studies will have to be carried out to confirm its use as a detection and prognostic tool for MM.

Conclusion

Mesothelioma is a devastating disease that has been causally linked to asbestos exposure. Although effort by several groups is in progress to understand this disease better so that more efficient therapies can be designed, there is still work to be done. The difficulty in diagnosing MM early and determining who is at risk of developing MM after asbestos exposure has hampered attempts at improving survival beyond 12 months after diagnoses. With the help of coordinated teams of researchers and physicians further progress in understanding this deadly disease and designing effective therapies can be achieved.