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Molecular pathways in malignant pleural mesothelioma
Cancer Letters 239 (2006) 183–189 www.elsevier.com/locate/canlet
Mini-review
Molecular pathways in malignant pleural mesothelioma Bryan A. Whitsona, Robert A. Kratzke* a
Division of Hematology/Oncology/Transplant University of Minnesota Medical School, Departments of Surgery, Section of Heme-Onc-Transplant, MMC 420, 480 Delaware St, SE, Minneapolis, MN 55455, USA b Division of Hematology/Oncology/Transplant University of Minnesota Medical School, Departments of Medicine, section of Heme-Onc-Transplant, MMC 420, 480 Delaware St, SE, Minneapolis, MN 55455, USA Received 1 August 2005; accepted 4 August 2005
Abstract Malignant pleural mesothelioma, although uncommon, is highly lethal. There is a high correlation between associated environmental exposure factors, carcinogens, and its development. Carcinogenesis is also mediated by genetic defects that result in loss of tumor suppressors or over expression of proto-oncogenes. Factors such as the loss of CDK inhibition function, IGF stimulatory pathways, p14ARF, p15INK4b, p16INK4a, p21, and p53 loss or mutation, VEGF and COX over expression are discussed. Correlations to potential therapeutic modalities are made. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Mesothelioma; CDK inhibitor; IGF
1. Introduction Malignant pleural mesothelioma is a disease that with a poor prognosis with a median survival time of 1 year [1]. The development of mesothelioma has been associated with asbestos exposure and potentially with SV40 tumor virus [2–5]. Identification of defects in critical molecular in mesothelioma has facilitated the understanding of the development and prognosis of mesothelioma. The limiting of carcinogen exposure, particularly to asbestos, and the continuing elucidation of the molecular pathways involved in
* Corresponding author. Tel.: C1 612 626 01234; fax: C1 612 625 6919. E-mail address: [email protected] (R.A. Kratzke).
transformation should lead to better disease control and greater therapeutic options in the coming decade.
2. Loss of CDK inhibitor function A genetic abnormality that recurs in the development of cancer is the loss of G1 to S checkpoint control locus [6]. An important moderator of this control is the presence of hypo-phosphorylation of pRb [7]. The phosphorylation of pRb is mediated by cyclin dependent kinases 4 and 6 (CDK4/6) with the corresponding inhibition of the kinase activity regulated by the family of CDK inhibitors. Among the most common deletions in mesothelioma, and other cancers, is at the 9p21 locus, the location encoding two critical CDK inhibitors p16INK4a and
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.08.010
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p15INK4b, as well as the p53 regulator p14ARF. In view of the high frequency of deletions at the 9p21 locus, it is not surprising that loss of expression of p16INK4a is associated with mesothelioma and is the most common acquired molecular lesion identified in this disease. The loss of the p16INK4a expression was found to be present in virtually all mesothelioma tumors and cell lines [7–9]. p14ARF is also encoded at the same locus. p14ARF rarely has independent loss of function from p16INK4a [9,10]. Although not independently deleted, there is a high association between p14ARF and p16INK4a both being simultaneously deleted [9]. The loss of p14ARF leads to destabilization of p53 and the subsequent increased malignant potential. The burden of asbestos exposure and the loss of p16 INK4a have been tied to mesothelioma presence. Potentially, the presence of the p16INK4a deletion allows for mesothelioma occurrence with less asbestos exposure and a lower number of packyear of smoking history [9]. As previously stated, not only is loss of p16INK4a a common result of deletion of 9p21, but in addition the homologous CDK inhibitor p15INK4b is absent in mesothelioma as well. p15INK4b has been found to be deleted in addition to p16INK4a in a high percentage of mesothelioma. Interestingly, the simultaneous deletion has been found to occur in cases where the 9p21 locus has not been deleted [11]. Nonetheless, no cases have been identified where loss of p15INK4b expression occurred without the corresponding loss of p16INK4a, while the inverse observation has been made arguing that the critical target of 9p21 deletion is loss of p16INK4a. Given that loss of p16INK4a is universally present in mesothelioma, reintroduction of this protein or analogous activity has a great potential for treatment. Experiments involving the re-introduction of p16INK4a into mesothelioma cell lines and xenografts have produced positive results. Mesothelioma cell culture was transfected with adenovirus expressing p16INK4a. The expression of p16INK4a correlated with decreased phosphorylation of pRb [12]. In a similar fashion to the re-expression of p16INK4a, the re-introduction of p14ARF by adenovirus vector has potential therapeutic benefit. After transduction of p14ARF, over expression of p14ARF in mesothelioma leads to increased levels of both p53 and p21. Additionally, there was an increase in Rb dephosphorylation. The presence of
functioning p53 and less phosphorylated Rb leads to G1 phase arrest and an inhibition in mesothelioma cell growth in culture [13].
3. IGF Among the multiple growth factors associated with tumor proliferation and migration is insulin-like growth factor (IGF) [14]. IGF can behave in an autocrine or paracrine fashion and stimulate either tumor growth or growth of adjacent tissues, respectively. The presence of IGF can also have stimulatory effects on extracellular matrix development [15]. Interestingly, the presence of a functioning IGF receptor is necessary for SV40 induced transformation of mesothelial cells arguing for an important role of the IGF axis in the development of mesothelioma [16]. Not surprisingly, expression of the two tyrosine kinase IGF receptors, IGF-R1 and IGF-R2, has been found to be present in all malignant mesothelioma cell lines [17]. This contribution of IGF pathways to mesothelioma tumor growth is further implicated by the ability of the inhibition of IGF receptor to decrease tumor growth in a hamster model [18]. In addition, the IGFR adaptor proteins insulin receptor substrate (IRS)-1 and -2 have been shown to play roles in the phenotype of disease in mesothelioma. Similar to what was previously seen in breast cancer, mesothelioma tumors that signal through IRS-1 are associated with increased cellular growth, while those that signal through IRS-2 are associated with increased cellular motility [19]. From this, it can be inferred that preferential utilization of either IRS-1 or IRS-2 may lead to different pathology. For example, a cell line with increased utilization of IRS-1 as the preferred IGFR adaptor protein could be expected to be more locally invasive, while a cell line with preferential signaling through IRS-2 could be more prone to metastasis. These observations lead to the potential therapeutic modalities in mesothelioma of targeted IGF receptor antagonists and/or inhibition of the downstream mediators of IGF. Additional members of the IGF axis include the IGFBP1-6 proteins that bind IGF and modulate the pathway. In surveys of mesothelioma, IGFBP 2, 4, and 5 all are found to be present while IGFBP 1, 3, and
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6 have been absent. The absence of the beneficial IGFBP-3 coupled with the presence of the deleterious IGFBP-4 would allow for a more aggressive phenotype [20]. This pattern, however, is not universal. In an array analysis with confirmatory polymerase chain reaction testing, IGF-1, IGF-II, IGF-R1 and IGF-R2 were found to be over-expressed in mesothelioma. IGF-1 was over-expressed 43.3 fold and the corresponding IGF receptor over 2.6 fold. In that series, IGFBP-1-4 were also over-expressed while IGFBP-5 was under-expressed [19]. This leads to the speculation that IGFBP-5 may be an inhibitor of IGF-R1 activation and that the decrease of the IGFBP5 allowed for over stimulation of the receptor allowing for potential autocrine stimulation [21]. These data, coupled with the correlation of the need of a functioning IGF receptor for SV40 induction, shed light on the aggressiveness of mesothelioma. However, the IGF pathway needs further, elucidation before the associations can be concreted.
4. p21, p27, p53 and Rb Mutations in p53 are among the most common acquired genetic lesions seen in cancers. Nonetheless p53 mutations are rarely seen in mesothelioma [10, 22–24]. However, this lack of gene mutations does not fully detail the effect of p53 on the natural history of p53. For example, p53 is inactivated by SV40 LT antigen and this possible r method of inactivating p53 may explain the absence of p53 mutations in mesothelioma [5] The SV40 LT tumor antigen binds to and inhibits both p53 and Rb proteins suggesting a potential carcinogenic pathway of SV40 LT in mesothelioma [3]. However, as previously discussed, loss of effective pRb function in mesothelioma already results from the universal deletion of p16INK4a presenting the compelling counterargument that SV40 LT is not necessary for inactivation of pRb. Loss of the same 9p21 locus also results in inactivation of p14ARF.and the consequent loss of p14ARF loss leads to Hdm2 mediated inactivation of p53. Thus the p53 activity is potentially lost in mesothelioma following the inactivation p14ARF associated with the common loss of 9p21 genetic material. The controversies that surround the SV40 hypothesis of mesothelioma carcinogenesis are
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summarized in this argument, namely that the simultaneous dual loss of p16INK4a and p14ARF argue for the inactivation of both p53 and pRb without SV40 expression necessitating an alternative role for SV40 in malignant transformation. The specific role of SV40 as an infectious cause of malignant mesothelioma remains in question. In a recent study, 69 human mesothelioma samples were evaluated through polyemerase chain reaction methods for SV40 DNA sequences. Primers specific to the SV40 large Tag region, which interacts with the pRb were used. In 100% of specimens these investigators were not able to detect SV40 large Tag sequences. The demographics of the patients were such that they were likely exposed to SV40 containing polio vaccines [25]. The lack of incoding in such a large number of mesotheliomas calls into question SV40’s true role in mesothelioma transformation. Nonetheless, a wide array of epidemiologic and biologic data support a role for SV40 in mesothelioma and the topic remains an area of intense controversy and investigation [3]. Not only are p53 and pRb important as immediate checkpoints but they also serve as checkpoints for downstream modulation. The best characterized downstream target of p53 activiation is the induction of p21 expression. p21, a multifunctional CDK inhibitor, plays a critical role in the G2/M cell cycle checkpoint. The expression of p21 highly associated with patient survival in mesothelioma. Increased expression of p21 is associated with patients having an improved survival in mesothelioma while decreased expression of p21 has been associated with decreased patient survival [26]. Restoring the p53-p21 axis of function is an additional potential therapeutic target in mesothelioma [4]. Immunohistochemical analyses have investigated the effects of p27 expression and the potential prognostic value in mesothelioma. p27, a cyclin E associated CDK inhibitor, has been implicated as a prognostic marker in several cancers including breast cancer. Similar to breast cancer, in mesothelioma low p27 expression has been correlated with a poor survival while high expression of p27 is associated longer survivals [27]. Expression of p27 is one of the better validated prognostic factors in patients with mesothelioma [28].
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5. BCL-2 Apoptosis is strongly mediated by the caspsase and bcl-2 pathways [29]. In particular, bcl-2 overexpression has been identified in many tumors and is associated with resistance to apoptosis. Similar to other tumors, the presence of bcl-2 over-expression has been identified in mesothelioma tumors [30]. Somewhat unexpectedly, lack of bcl-2 expression was associated with a higher apoptotic index in mesothelioma which correlated with poor survival [31]. Whether bcl-2 expression can be a clinically useful prognostic marker in mesothelioma is not clear. Additionally, the presence of bcl-2 expression has been used as a diagnostic tool as well. The absence of the expression of bcl-2 can be helpful in the discrimination between mesothelioma and reactive cells [32].
6. VEGF and COX-2 A common component to all malignancy is the ability to develop vascular supply to support tumor growth. Vascular Endothelial Growth Factor (VEGF) and its associated receptor family play a significant role in mediating this process. As malignancies grow, they have an increased metabolic demand and need for vascular delivery. In this setting, VEGF stimulates the growth of new blood vessels. Not surprisingly, VEGF is influential in mesothelioma as well. Interestingly, VEGF over-expression is reported to be stimulated by SV40 infection. VEGF expression is not only implicated in angiogenesis, but in growth factor stimulation as well [33]. As the tumor develops the capacity to form new blood vessels there is a concurrent increase in growth factor secretion associated with increased malignant potential. Interest in the role of over-expression of cyclooxygenase-2 (COX-2) and its involvement in tumorigenesis are growing. There are increasing data to show that COX-2 plays a role in mesothelioma pathology. For example, COX-2 over-expression is associated with a poor prognosis in mesothelioma. Multivariate analysis of p21, p53 and COX-2 expression have shown COX-2 expression to be prognostic [34]. COX-2 over expression in and of itself, is associated with poor prognosis [35].
Utilization of COX-2 inhibitors may play a potent role in multimodal chemotherapeutic approaches to the treatment of mesothelioma.
7. EGFR expression Epidermal growth factor receptor (EGFR) has been found to be over-expressed in malignant mesothelioma. The over-expression of EGFR has been associated with increased proliferation and angiogenesis. Gefitinib, a selective inhibitor of EGFR, a receptor tyrosine kinase, has been developed and has entered clinical use. After studies demonstrated that mesothelioma treated with gefitinib had inhibited proliferation while apoptosis increased in mesothelioma cell lines, a phase II study of its effectiveness in humans was undertaken. The EGFR receptor was found to be over-expressed in almost all patients (97.6%), with heavy over-expression identified as a favorable prognostic factor. However, the treatment of the patients with gefitinib had no effect on the failure free survival, but the overall survival was more than doubled, from 3.64 to 8.1 months [36].
8. NF2, RASSF1A, and WT1 Population studies have linked the autosomally dominant disease neurofibromatosis type 2 (NF2), to the loss of the function of the gene located on the long arm of chromosome 22 [37,38], the so called schwannomin protein or merlin (meosin ezrin radixin-like protein). In malignant mesothelioma, the loss of chromosome 22 gene products is an extremely common, recurring occurrence [39]. Analysis of mesothelioma cell lines through protein coding sequencing has revealed that 100% (8/8) of cell lines which had single strand conformation polymorphisms (SSCP) of cDNA (8/15 or 53%) demonstrated high levels of mutations and truncation of merlin [40]. A growing body of literature is reinforcing the concept that the NF2 gene acts as a tumor suppressor gene in mesothelioma and that the mutation of the NF2 gene is a highly common occurrence in the malignant phenotype. Hypermethylation of DNA is significant in gene suppression in malignancy. In a series of 66
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mesothelioma samples, 32% demonstrated hypermethylation of the RASSF1A tumor suppressor gene candidate [41]. This gene maps to a site which is common for loss of heterozygosity in cancers, the 3p21.3 locus. In this study, 52% of the tumors analyzed contined SV40 Tag sequences with a high methylation index [41]. A link between the RASSF1A tumor suppressor gene inactivation, SV40 infection, and malignant mesothelioma may be playing a role in transformation. Work in pediatric nephroblastomas, also known as Wilms Tumor, has been able to identify a suppressor gene, Wilms Tumor Susceptibility Gene 1 (WT1) [42]. The absence of WT1 function, either though gene loss or inactivation, is present in most pediatric nephroblastomas. After normal WT1 expression was demonstrated in normal mesothelium and urothelium, mutations of WT1 were demonstrated in mesothelioma [43]. Though immunohistochemistry, WT1 mutations were found to be overexpressed in malignant mesothelioma [44,45]. WT1 identification, through nuclear staining, has been identified in 75– 100% of mesothelioma tumor and cell lines. Overexpression of WT1 is rare in lung cancer. As a diagnostic tool, WT1 expression use has grown in utility for identification of and distinguishing mesothelioma from lung cancer [44].
9. Conclusions Mesothelioma is associated with environmental exposure to asbestos and potentially with SV40 infection. There are many genetic defects that contribute to the outcome of mesothelioma. Some of the most common are associated with deletion at the 9p21 locus and consequent loss of p15INK4b, p16INK4a, and p14ARF. Identification and quantification of WT1 expression can have diagnostic benefit in histologically difficult tumors. Increasingly, the IGF axis is also being found to be an important regulator of mesothelioma growth and tumorigenesis. This stimulatory pathway leads to increased proliferation and migration. The angiogenic properties accompanying over-expression of both VEGF and COX-2 are of increasing interest not only in mesothelioma, but in other malignancies as well. Inhibition of RASSF1A hypermethylation is a potential avenue of therapy. Novel chemotherapeutic
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agents that selectively inhibit these growth factor pathways could play an important role in combined multimodal therapy in curing this deadly disease.
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Mini-review
Molecular pathways in malignant pleural mesothelioma Bryan A. Whitsona, Robert A. Kratzke* a
Division of Hematology/Oncology/Transplant University of Minnesota Medical School, Departments of Surgery, Section of Heme-Onc-Transplant, MMC 420, 480 Delaware St, SE, Minneapolis, MN 55455, USA b Division of Hematology/Oncology/Transplant University of Minnesota Medical School, Departments of Medicine, section of Heme-Onc-Transplant, MMC 420, 480 Delaware St, SE, Minneapolis, MN 55455, USA Received 1 August 2005; accepted 4 August 2005
Abstract Malignant pleural mesothelioma, although uncommon, is highly lethal. There is a high correlation between associated environmental exposure factors, carcinogens, and its development. Carcinogenesis is also mediated by genetic defects that result in loss of tumor suppressors or over expression of proto-oncogenes. Factors such as the loss of CDK inhibition function, IGF stimulatory pathways, p14ARF, p15INK4b, p16INK4a, p21, and p53 loss or mutation, VEGF and COX over expression are discussed. Correlations to potential therapeutic modalities are made. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Mesothelioma; CDK inhibitor; IGF
1. Introduction Malignant pleural mesothelioma is a disease that with a poor prognosis with a median survival time of 1 year [1]. The development of mesothelioma has been associated with asbestos exposure and potentially with SV40 tumor virus [2–5]. Identification of defects in critical molecular in mesothelioma has facilitated the understanding of the development and prognosis of mesothelioma. The limiting of carcinogen exposure, particularly to asbestos, and the continuing elucidation of the molecular pathways involved in
* Corresponding author. Tel.: C1 612 626 01234; fax: C1 612 625 6919. E-mail address: [email protected] (R.A. Kratzke).
transformation should lead to better disease control and greater therapeutic options in the coming decade.
2. Loss of CDK inhibitor function A genetic abnormality that recurs in the development of cancer is the loss of G1 to S checkpoint control locus [6]. An important moderator of this control is the presence of hypo-phosphorylation of pRb [7]. The phosphorylation of pRb is mediated by cyclin dependent kinases 4 and 6 (CDK4/6) with the corresponding inhibition of the kinase activity regulated by the family of CDK inhibitors. Among the most common deletions in mesothelioma, and other cancers, is at the 9p21 locus, the location encoding two critical CDK inhibitors p16INK4a and
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.08.010
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p15INK4b, as well as the p53 regulator p14ARF. In view of the high frequency of deletions at the 9p21 locus, it is not surprising that loss of expression of p16INK4a is associated with mesothelioma and is the most common acquired molecular lesion identified in this disease. The loss of the p16INK4a expression was found to be present in virtually all mesothelioma tumors and cell lines [7–9]. p14ARF is also encoded at the same locus. p14ARF rarely has independent loss of function from p16INK4a [9,10]. Although not independently deleted, there is a high association between p14ARF and p16INK4a both being simultaneously deleted [9]. The loss of p14ARF leads to destabilization of p53 and the subsequent increased malignant potential. The burden of asbestos exposure and the loss of p16 INK4a have been tied to mesothelioma presence. Potentially, the presence of the p16INK4a deletion allows for mesothelioma occurrence with less asbestos exposure and a lower number of packyear of smoking history [9]. As previously stated, not only is loss of p16INK4a a common result of deletion of 9p21, but in addition the homologous CDK inhibitor p15INK4b is absent in mesothelioma as well. p15INK4b has been found to be deleted in addition to p16INK4a in a high percentage of mesothelioma. Interestingly, the simultaneous deletion has been found to occur in cases where the 9p21 locus has not been deleted [11]. Nonetheless, no cases have been identified where loss of p15INK4b expression occurred without the corresponding loss of p16INK4a, while the inverse observation has been made arguing that the critical target of 9p21 deletion is loss of p16INK4a. Given that loss of p16INK4a is universally present in mesothelioma, reintroduction of this protein or analogous activity has a great potential for treatment. Experiments involving the re-introduction of p16INK4a into mesothelioma cell lines and xenografts have produced positive results. Mesothelioma cell culture was transfected with adenovirus expressing p16INK4a. The expression of p16INK4a correlated with decreased phosphorylation of pRb [12]. In a similar fashion to the re-expression of p16INK4a, the re-introduction of p14ARF by adenovirus vector has potential therapeutic benefit. After transduction of p14ARF, over expression of p14ARF in mesothelioma leads to increased levels of both p53 and p21. Additionally, there was an increase in Rb dephosphorylation. The presence of
functioning p53 and less phosphorylated Rb leads to G1 phase arrest and an inhibition in mesothelioma cell growth in culture [13].
3. IGF Among the multiple growth factors associated with tumor proliferation and migration is insulin-like growth factor (IGF) [14]. IGF can behave in an autocrine or paracrine fashion and stimulate either tumor growth or growth of adjacent tissues, respectively. The presence of IGF can also have stimulatory effects on extracellular matrix development [15]. Interestingly, the presence of a functioning IGF receptor is necessary for SV40 induced transformation of mesothelial cells arguing for an important role of the IGF axis in the development of mesothelioma [16]. Not surprisingly, expression of the two tyrosine kinase IGF receptors, IGF-R1 and IGF-R2, has been found to be present in all malignant mesothelioma cell lines [17]. This contribution of IGF pathways to mesothelioma tumor growth is further implicated by the ability of the inhibition of IGF receptor to decrease tumor growth in a hamster model [18]. In addition, the IGFR adaptor proteins insulin receptor substrate (IRS)-1 and -2 have been shown to play roles in the phenotype of disease in mesothelioma. Similar to what was previously seen in breast cancer, mesothelioma tumors that signal through IRS-1 are associated with increased cellular growth, while those that signal through IRS-2 are associated with increased cellular motility [19]. From this, it can be inferred that preferential utilization of either IRS-1 or IRS-2 may lead to different pathology. For example, a cell line with increased utilization of IRS-1 as the preferred IGFR adaptor protein could be expected to be more locally invasive, while a cell line with preferential signaling through IRS-2 could be more prone to metastasis. These observations lead to the potential therapeutic modalities in mesothelioma of targeted IGF receptor antagonists and/or inhibition of the downstream mediators of IGF. Additional members of the IGF axis include the IGFBP1-6 proteins that bind IGF and modulate the pathway. In surveys of mesothelioma, IGFBP 2, 4, and 5 all are found to be present while IGFBP 1, 3, and
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6 have been absent. The absence of the beneficial IGFBP-3 coupled with the presence of the deleterious IGFBP-4 would allow for a more aggressive phenotype [20]. This pattern, however, is not universal. In an array analysis with confirmatory polymerase chain reaction testing, IGF-1, IGF-II, IGF-R1 and IGF-R2 were found to be over-expressed in mesothelioma. IGF-1 was over-expressed 43.3 fold and the corresponding IGF receptor over 2.6 fold. In that series, IGFBP-1-4 were also over-expressed while IGFBP-5 was under-expressed [19]. This leads to the speculation that IGFBP-5 may be an inhibitor of IGF-R1 activation and that the decrease of the IGFBP5 allowed for over stimulation of the receptor allowing for potential autocrine stimulation [21]. These data, coupled with the correlation of the need of a functioning IGF receptor for SV40 induction, shed light on the aggressiveness of mesothelioma. However, the IGF pathway needs further, elucidation before the associations can be concreted.
4. p21, p27, p53 and Rb Mutations in p53 are among the most common acquired genetic lesions seen in cancers. Nonetheless p53 mutations are rarely seen in mesothelioma [10, 22–24]. However, this lack of gene mutations does not fully detail the effect of p53 on the natural history of p53. For example, p53 is inactivated by SV40 LT antigen and this possible r method of inactivating p53 may explain the absence of p53 mutations in mesothelioma [5] The SV40 LT tumor antigen binds to and inhibits both p53 and Rb proteins suggesting a potential carcinogenic pathway of SV40 LT in mesothelioma [3]. However, as previously discussed, loss of effective pRb function in mesothelioma already results from the universal deletion of p16INK4a presenting the compelling counterargument that SV40 LT is not necessary for inactivation of pRb. Loss of the same 9p21 locus also results in inactivation of p14ARF.and the consequent loss of p14ARF loss leads to Hdm2 mediated inactivation of p53. Thus the p53 activity is potentially lost in mesothelioma following the inactivation p14ARF associated with the common loss of 9p21 genetic material. The controversies that surround the SV40 hypothesis of mesothelioma carcinogenesis are
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summarized in this argument, namely that the simultaneous dual loss of p16INK4a and p14ARF argue for the inactivation of both p53 and pRb without SV40 expression necessitating an alternative role for SV40 in malignant transformation. The specific role of SV40 as an infectious cause of malignant mesothelioma remains in question. In a recent study, 69 human mesothelioma samples were evaluated through polyemerase chain reaction methods for SV40 DNA sequences. Primers specific to the SV40 large Tag region, which interacts with the pRb were used. In 100% of specimens these investigators were not able to detect SV40 large Tag sequences. The demographics of the patients were such that they were likely exposed to SV40 containing polio vaccines [25]. The lack of incoding in such a large number of mesotheliomas calls into question SV40’s true role in mesothelioma transformation. Nonetheless, a wide array of epidemiologic and biologic data support a role for SV40 in mesothelioma and the topic remains an area of intense controversy and investigation [3]. Not only are p53 and pRb important as immediate checkpoints but they also serve as checkpoints for downstream modulation. The best characterized downstream target of p53 activiation is the induction of p21 expression. p21, a multifunctional CDK inhibitor, plays a critical role in the G2/M cell cycle checkpoint. The expression of p21 highly associated with patient survival in mesothelioma. Increased expression of p21 is associated with patients having an improved survival in mesothelioma while decreased expression of p21 has been associated with decreased patient survival [26]. Restoring the p53-p21 axis of function is an additional potential therapeutic target in mesothelioma [4]. Immunohistochemical analyses have investigated the effects of p27 expression and the potential prognostic value in mesothelioma. p27, a cyclin E associated CDK inhibitor, has been implicated as a prognostic marker in several cancers including breast cancer. Similar to breast cancer, in mesothelioma low p27 expression has been correlated with a poor survival while high expression of p27 is associated longer survivals [27]. Expression of p27 is one of the better validated prognostic factors in patients with mesothelioma [28].
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5. BCL-2 Apoptosis is strongly mediated by the caspsase and bcl-2 pathways [29]. In particular, bcl-2 overexpression has been identified in many tumors and is associated with resistance to apoptosis. Similar to other tumors, the presence of bcl-2 over-expression has been identified in mesothelioma tumors [30]. Somewhat unexpectedly, lack of bcl-2 expression was associated with a higher apoptotic index in mesothelioma which correlated with poor survival [31]. Whether bcl-2 expression can be a clinically useful prognostic marker in mesothelioma is not clear. Additionally, the presence of bcl-2 expression has been used as a diagnostic tool as well. The absence of the expression of bcl-2 can be helpful in the discrimination between mesothelioma and reactive cells [32].
6. VEGF and COX-2 A common component to all malignancy is the ability to develop vascular supply to support tumor growth. Vascular Endothelial Growth Factor (VEGF) and its associated receptor family play a significant role in mediating this process. As malignancies grow, they have an increased metabolic demand and need for vascular delivery. In this setting, VEGF stimulates the growth of new blood vessels. Not surprisingly, VEGF is influential in mesothelioma as well. Interestingly, VEGF over-expression is reported to be stimulated by SV40 infection. VEGF expression is not only implicated in angiogenesis, but in growth factor stimulation as well [33]. As the tumor develops the capacity to form new blood vessels there is a concurrent increase in growth factor secretion associated with increased malignant potential. Interest in the role of over-expression of cyclooxygenase-2 (COX-2) and its involvement in tumorigenesis are growing. There are increasing data to show that COX-2 plays a role in mesothelioma pathology. For example, COX-2 over-expression is associated with a poor prognosis in mesothelioma. Multivariate analysis of p21, p53 and COX-2 expression have shown COX-2 expression to be prognostic [34]. COX-2 over expression in and of itself, is associated with poor prognosis [35].
Utilization of COX-2 inhibitors may play a potent role in multimodal chemotherapeutic approaches to the treatment of mesothelioma.
7. EGFR expression Epidermal growth factor receptor (EGFR) has been found to be over-expressed in malignant mesothelioma. The over-expression of EGFR has been associated with increased proliferation and angiogenesis. Gefitinib, a selective inhibitor of EGFR, a receptor tyrosine kinase, has been developed and has entered clinical use. After studies demonstrated that mesothelioma treated with gefitinib had inhibited proliferation while apoptosis increased in mesothelioma cell lines, a phase II study of its effectiveness in humans was undertaken. The EGFR receptor was found to be over-expressed in almost all patients (97.6%), with heavy over-expression identified as a favorable prognostic factor. However, the treatment of the patients with gefitinib had no effect on the failure free survival, but the overall survival was more than doubled, from 3.64 to 8.1 months [36].
8. NF2, RASSF1A, and WT1 Population studies have linked the autosomally dominant disease neurofibromatosis type 2 (NF2), to the loss of the function of the gene located on the long arm of chromosome 22 [37,38], the so called schwannomin protein or merlin (meosin ezrin radixin-like protein). In malignant mesothelioma, the loss of chromosome 22 gene products is an extremely common, recurring occurrence [39]. Analysis of mesothelioma cell lines through protein coding sequencing has revealed that 100% (8/8) of cell lines which had single strand conformation polymorphisms (SSCP) of cDNA (8/15 or 53%) demonstrated high levels of mutations and truncation of merlin [40]. A growing body of literature is reinforcing the concept that the NF2 gene acts as a tumor suppressor gene in mesothelioma and that the mutation of the NF2 gene is a highly common occurrence in the malignant phenotype. Hypermethylation of DNA is significant in gene suppression in malignancy. In a series of 66
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mesothelioma samples, 32% demonstrated hypermethylation of the RASSF1A tumor suppressor gene candidate [41]. This gene maps to a site which is common for loss of heterozygosity in cancers, the 3p21.3 locus. In this study, 52% of the tumors analyzed contined SV40 Tag sequences with a high methylation index [41]. A link between the RASSF1A tumor suppressor gene inactivation, SV40 infection, and malignant mesothelioma may be playing a role in transformation. Work in pediatric nephroblastomas, also known as Wilms Tumor, has been able to identify a suppressor gene, Wilms Tumor Susceptibility Gene 1 (WT1) [42]. The absence of WT1 function, either though gene loss or inactivation, is present in most pediatric nephroblastomas. After normal WT1 expression was demonstrated in normal mesothelium and urothelium, mutations of WT1 were demonstrated in mesothelioma [43]. Though immunohistochemistry, WT1 mutations were found to be overexpressed in malignant mesothelioma [44,45]. WT1 identification, through nuclear staining, has been identified in 75– 100% of mesothelioma tumor and cell lines. Overexpression of WT1 is rare in lung cancer. As a diagnostic tool, WT1 expression use has grown in utility for identification of and distinguishing mesothelioma from lung cancer [44].
9. Conclusions Mesothelioma is associated with environmental exposure to asbestos and potentially with SV40 infection. There are many genetic defects that contribute to the outcome of mesothelioma. Some of the most common are associated with deletion at the 9p21 locus and consequent loss of p15INK4b, p16INK4a, and p14ARF. Identification and quantification of WT1 expression can have diagnostic benefit in histologically difficult tumors. Increasingly, the IGF axis is also being found to be an important regulator of mesothelioma growth and tumorigenesis. This stimulatory pathway leads to increased proliferation and migration. The angiogenic properties accompanying over-expression of both VEGF and COX-2 are of increasing interest not only in mesothelioma, but in other malignancies as well. Inhibition of RASSF1A hypermethylation is a potential avenue of therapy. Novel chemotherapeutic
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agents that selectively inhibit these growth factor pathways could play an important role in combined multimodal therapy in curing this deadly disease.
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