and Environment, Vol. 33, No. 1 pp. 4–9 (2011)

Review Elucidation of Asbestos-induced Mesothelial Carcinogenesis toward Its Prevention

Li Jiang and Shinya Toyokuni1 Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan

(Received November 10, 2010; Revised December 29, 2010; Accepted January 4, 2011)

Human exposure to asbestos ˆbers has been associated dily due to its long latency period (2). Moreover, there is with diŠuse malignant mesothelioma (DMM) in the pleural also continuous use of asbestos in some developing and abdominal cavity. Despite advancements in the countries such as China and India. Thus, it is predicted molecular analyses of human cases of DMM and animal that the incidence of malignant mesothelioma will con- models, the understanding of the carcinogenic mechan- tinue to rise in the future. For instance, it is estimated isms remains still limited. There are basically three that in Japan alone, mesothelioma incidence will peak hypotheses regarding the pathogenesis of asbestos-in- in 2025 with a cumulative 100,000 deaths (3). Malignant duced DMM, which may be integrated as follows; (1) the ``oxidative stress theory'' is based on the fact that phago- mesothelioma is a highly aggressive tumor that shows cytic cells that engulf asbestos ˆbers produce large resistance to the currently available therapeutic modali- amounts of reactive oxygen species (ROS) due to their ties. Hence, the elucidation of the molecular mechan- inability to digest the ˆbers, and that iron contained in isms of asbestos-induced mesothelioma development is crocidolite and amosite ˆbers works as a catalyst for the of the utmost importance at the moment in order to de- generation of ROS, (2) the `` tangling theory'' signthemosteŠectivepreventiveandtherapeuticap- postulates that asbestos ˆbers impair the equivalent distri- proaches for malignant mesothelioma. bution of during mitosis, and (3) the ``theory of adsorbing many speciˆc as well as carcinogen- Types of Asbestos Fibers ic molecules'' states that asbestos ˆbers in vivo concen- Asbestos is a naturally occurring silicate mineral. The trate speciˆc proteins or chemicals including the compo- main characteristic of this mineral is that it exists in ˆ- nents of cigarette smoke and radioactive chemical ele- ment. Recent studies suggest that local iron overload is a brous form. Commercial use of asbestos has a long key event. Elucidation of the major mechanisms underlying history dating back to centuries ago. Its several phys- DMM would be helpful for the development of strategies icochemical properties, such as durability, heat- and to prevent DMM generation in people who have been ex- chemical-resistance, and tensile strength, made it an ex- posed to asbestos. traordinary material for various industrial applications. It was even once considered to be a miraculous mineral Key words: asbestos, mesothelioma, iron, oxidative stress due to its widespread applicability. Some examples of products in which asbestos ˆbers were incorporated in- clude heat insulators, pipes, fabrics and automobile Introduction brakes. Asbestos was mined extensively especially dur- Asbestos exposure has been established as a primary ing the World War II period when it was mainly used factor contributing to the development of malignant for building ships (1). mesothelioma (1). Mesothelioma develops from Asbestos is a general name designated for a group of mesothelial cells, which line the pleural, peritoneal and ˆbrous minerals that can be divided into two major pericardial cavities. Malignant pleural mesothelioma oc- classes, known as serpentines and amphiboles, respec- curs most frequently in human cases. The latency period tively. Under serpentines, there is only one member of malignant mesothelioma development after the ˆrst known as chrysotile (white asbestos) while under amphi- asbestos exposure averages around 30–40 years. Cur- boles, there are ˆve members known as crocidolite (blue rently, it is reported that approximately 2,500–3,000 asbestos), amosite (brown asbestos), tremolite, an- new cases of mesothelioma are diagnosed in the United 1 States every year. While in Japan, more than 1,000 cases Correspondence to: Shinya Toyokuni, Department of Pathology and Biological Responses, Nagoya University Graduate School of Medi- of mesothelioma are diagnosed each year. In spite of the cine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan. fact that asbestos use has been banned in many coun- Tel: ＋81-52-744-2086, Fax: ＋81-52-744-2091, E-mail: toyokuni＠ tries, recently mesothelioma incidence is increasing stea- med.nagoya-u.ac.jp

 The Japanese Environmental Mutagen Society 4 Asbestos-induced Mesothelial Carcinogenesis thophyllite and actinolite. Among these diŠerent as- ated involves the macrophages. Asbestos ˆbers, when bestos types, chrysotile has the most widespread usage inhaled and deposited within the tissues, act as foreign followed by crocidolite and amosite. The other three as- particles that are able to trigger in‰ammatory response. bestos types have minimal industrial usage (4). Morpho- Macrophages, which are recruited to the area of as- logically, chrysotile has a curly appearance while mem- bestos deposition, attempt to phagocytose the asbestos bers of the amphibole family have a straight, rod-like ˆbers but might fail to do so because of the existence of appearance. exceedingly long ˆbers (7). Under these conditions known as the ``frustrated phagocytosis'', large amounts DiŠerent Theories of Asbestos-induced of reactive oxygen species (ROS) are released by the Pathogenesis macrophages. Our laboratory has recently demonstrat- Several diŠerent theories have been proposed to ac- ed that asbestos ˆbers are able to induce the production count for the asbestos-induced carcinogenesis (Fig. of ROS and these ROS in turn are able to cause DNA 1A-C). The ˆrst theory is known as the ``oxidative stress damage (8). theory.'' As implied by the name, this theory postulates Another theory proposed to be responsible for that asbestos ˆbers are able to induce mesothelioma de- asbestos-induced carcinogenesis is known as the ``chro- velopment through their ability to generate oxidative mosome tangling theory.'' According to this theory, as- stress (5). According to this theory, oxidative stress can bestos ˆbers are able to physically disrupt chromosomal be generated via two means. Two (crocidolite and amo- structure, especially during cell division. Chromosomal site) of the most widely used asbestos ˆbers contain iron structure disruption during mitosis can lead to in- as one of their chemical components (¿30z)anditwas heritance of abnormal chromosomes by the daughter suggested that these iron can act as a catalyst to enhance cells. Indeed, we have recently demonstrated that as- the production of highly reactive hydroxyl radicals bestos ˆbers were actively taken up by cultured cells and through the well-known Fenton reaction (6). Another these ˆbers traveled through the cytoplasm and even- mechanism through which oxidative stress can be gener- tually entered into the nuclear area during mitosis,

Fig. 1. Hypothesized mechanisms of asbestos-induced mesothelial carcinogenesis. A. Oxidative stress theory consists of two diŠerent mechan- isms: 1) asbestos ˆber itself works as a catalyst in the case of crocidolite or amosite; 2) macrophages that engulfed extremely long ˆbrous foreign materials are frustrated because they can neither carry them away to lymph nodes nor digest them. B. Chromosome tangling theory postulates the interaction of asbestos ˆber actively engulfed by mesothelial cell and chromosome at mitosis. C. Adsorption theory suggests the surface adsorptive activity of asbestos ˆber to proteins, nucleic acids and exogenous carcinogenic molecules. Refer to text for details.

5 Li Jiang and Shinya Toyokuni where they can directly interact with the chromosomes strand breaks, thereby predisposing to mesothelioma (8). Thus, our observation further supports the concept development via its clastogenicity. It is recently recog- of chromosome tangling theory. nized that iron overload in general is associated with In addition to these two theories, another well-accept- carcinogenesis (12). ed theory of asbestos-induced carcinogenesis is known as the ``adsorption theory.'' This theory suggests that Genomic Alterations in Malignant Mesothelioma the surface of asbestos ˆber of each kind has a high Like any other types of cancer, some forms of genetic a‹nity for certain speciˆc proteins and molecules (9). changes also take place during the development of This is not only due to the presence of positive and/or malignant mesothelioma. However, unlike most of the negative charges on the surface of asbestos ˆbers but other types of cancer in which alterations involving also due to its physical characteristics. As a conse- genes such as p53 and ras family are very frequent, quence, various molecules, including those with car- malignant mesothelioma generally displays a distinct cinogenic property are adsorbed on the surface of as- spectrum of genetic alterations that most probably bestos ˆbers and accumulated there. For instance, as- results from its unique impact of asbestos exposure. bestos ˆbers have been reported to be able to bind to Many previous reports show in agreement that malig- carcinogenic molecules contained in cigarette smoke nant mesothelioma is associated with an extensive (10) and radium (11). We believe that these three genomic instability, which includes aneuploidy and mechanisms coexist during asbestos-induced mesotheli- structural rearrangement of various chromosomes (13). al carcinogenesis and even interact each other. For ex- Results obtained from chromosome banding techniques ample, chrysotile adsorbs hemoglobin after inducing revealed remarkably complex karyotypes in malignant hemolysis, leading to generation of hydroxyl radical via mesothelioma cases. The most consistent chromosomal heme iron. This kind of oxidative stress may cause ox- alterations were observed in the chromosomes 3p, 4, 6q idative DNA damage of the genome since asbestos ˆbers and 9p as well as chromosome 13q. In addition to these, have an a‹nity for actin and histones (Nagai H and complete loss of one of the two copies of chromosome Toyokuni S, unpublished observations). 22 was also found to take place frequently in malignant mesothelioma (14,15). Such chromosomal abnormali- Involvement of Iron in Asbestos-induced ties have been associated with loss of tumor suppressor Carcinogenesis genes. As mentioned earlier, chrysotile, crocidolite and amo- The tumor suppressor p16INK4a (mapped to 9p21 lo- site ˆbers are among the most commonly used asbestos cus), whose product is a cyclin-dependent kinase inhibi- types. As a result, most research studies have focused on tor, is the most frequently inactivated tumor suppressor these three asbestos types compared to the other three encountered in malignant mesothelioma. This ˆnd- asbestos ˆbers. Chemical composition analysis revealed ing underlies a signiˆcant diŠerence of malignant that both crocidolite and amosite have a high content of mesothelioma from other types of cancer, in which p53 iron. In contrast, chrysotile does not contain any iron in inactivation is found to be more predominant. Inactiva- its content but subtle amount of iron is present as a con- tion of p16INK4a signiˆes a loss of cell cycle regulation as taminant metal (1). Our laboratory has demonstrated p16INK4a normally regulates the progression of cell cycle that asbestos ˆbers are able to generate free radicals that through the cyclin-dependent kinase 4/cyclin D/RB contribute to the buildup of oxidative stress. By using pathway (Fig. 2A). Inactivation of p16INK4a via epigenet- the electron spin resonance determination, we were able ic mechanisms is less common in malignant mesothelio- to show in in vitro experiments that crocidolite and macasescomparedtoothertypesofmalignancy.In- amosite ˆbers generated a large amount of hydroxyl stead, homozygous deletion of p16INK4a is detected at a radicals in the presence of 1 mM H2O2 (8). Among these very high frequency in malignant mesothelioma. Results three ˆbers, amosite has the highest ability to generate from previous studies have shown that the majority of free radicals followed by crocidolite. When we added primary mesothelioma tumors and mesothelioma cell nitrilotriacetic acid (NTA), an iron chelator to promote lines were deleted for their p16INK4a in a homozygous the Fenton reaction, to the asbestos ˆbers, we found a manner (16). Deletion of this region involves simultane- signiˆcant increase in hydroxyl radical generation by the ous deletion of P14 (ARF) , a product by alter- asbestos ˆbers. The higher potential of amosite and native splicing of p16INK4a, leading eventually to loss of crocidolite ˆbers in the generation of free radicals corre- TP53 and apoptosis-resistance (17,18) (Fig. 2A). We be- lates proportionally with their iron content. Our ˆnd- lieve that this is one of the most important parts of the ings thus indicate that iron contained in the asbestos genome and also the major target in iron overload-in- ˆbers plays a crucial role in the induction of oxidative duced carcinogenesis (19,20). stress. We further showed that the free radicals generat- As noted earlier, loss of chromosome 22 is also fre- ed by asbestos ˆbers were able to cause DNA double- quently found in malignant mesothelioma. Positional

6 Asbestos-induced Mesothelial Carcinogenesis

Fig. 2. Important signaling pathways that are either activated or inactivated in malignant mesothelioma (MM). A. Homozygous deletion of CDKN2A (p16INK4a) tumor suppressor gene is the most common genetic alteration in human and rat malignant mesothelioma. This alteration inac- tivates both retinoblastoma and p53 pathway and promote cellular proliferation while preventing apoptosis. B. Activation of YAP (yes-associated protein) occurs frequently in malignant mesothelioma either by genomic ampliˆcation or its dephophorylation. Phosphorylation of YAP is regu- lated by HIPPO pathway consisting of Merlin (NF2), SAV, MST and LATS. C. WT1 works here as an oncogenic protein, leading to cellular proliferation whereas BASP1 and phosphorylated WT1 (WT1P) antagonize the oncogenic function. Refer to text for details.

cloning subsequently identiˆed a tumor suppressor Another gene consistently mutated in malignant gene, known as neuroˆbromatosis type 2 (NF2)geneat mesothelioma is known as the Wilm's tumor gene the 22q12 locus. It was reported that the NF2 gene is in- (WT1). WT1 gene was originally isolated as a tumor activated in 40–50z of malignant mesothelioma. Inacti- suppressor gene and was observed to be inactivated in a vation of NF2 can occur through homozygous deletion, subset of Wilm's tumor. However, it was later found to nonsense mutation or missense mutation (21,22). be overexpressed in several types of cancer including NF2＋/－ mice have been shown to be more susceptible to lung cancer, leukemia, breast cancer and thyroid can- mesothelioma development after asbestos exposure cer. Thus, it was proposed that the WT1 gene more (23). The NF2 gene encodes a 70 kD protein known as likely plays an oncogenic role rather than tumor sup- Schwannomin or Merlin. Merlin is one of the compo- pression in most types of cancer. Likewise, WT1 is nents of the mammalian Hippo signaling cascade. Other overexpressed in malignant mesothelioma. Immuno- components include WW45, MST, LATS and YAP histochemical analysis clearly demonstrated nuclear (24–26). Recent studies suggest that YAP, which func- staining for WT1 in 75–100z of primary mesothelioma tions as a transcriptional co-activator, plays a crucial tumors and mesothelioma cell lines examined (27–29). role in the proliferation and survival of mesothelioma The WT1 gene encodes for a zinc ˆnger transcription cells. Merlin, on the other hand, exerts its tumor sup- factor which activates the transcription of various pressive eŠect via phosphorylation and subsequent growth factors and growth factor receptors (Fig. 2C). translocation of YAP from the nucleus into the cytoplasm, thus inhibiting the tumor-promoting activity Conclusion of YAP. Loss of the NF2 gene therefore has a great Asbestos-induced malignant mesothelioma develop- positive impact on the development of malignant ment most probably involves the chronic generation of mesothelioma (Fig. 2B). oxidative stress. The ability of asbestos ˆbers to gener-

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