Cell-Cycle Molecules in Mesothelioma: an Overview

J. Exp. Clin. Cancer Res., 26,4, 2007 Review Cell-Cycle Molecules in Mesothelioma: an Overview E. P. Spugnini 1, M. Campioni 2, A. D'Avino 2, G. Caruso 2, G. Citro 1, A. Baldi 2 1 SAFU Department, Regina Elena Cancer Institute, Rome; 2 Dept. Biochemistry, Sect. Pathology, 2nd University of Naples, Naples - Italy Cell cycle progression is mediated by a group of proteins named cyclins that activate a highly conserved family of protein kinases, the cyclin-dependent kinases (CDKs). CDKs are also regulated by related proteins called cdk inhibitors, grouped into two families: the INK4 inhibitors (p16, p15, p19 and p18) and the Cip/Kip inhibitors (p21, p27). Moreover, several tumour suppressor genes (such as Retinoblastoma gene and p53 gene) are implicated in the regulation of the molecular mechanism of cell division. Several studies report the importance of cell cycle regulator proteins in the pathogenesis and the prognosis of mesothelioma. This article will review the most recent data from the literature about the expression and the diagnostic and prognostic significance of cell cycle molecules in mesothelioma. Key Words: INK4 inhibitors, p21, p53, Rb2, Mesothelioma Malignant mesothelioma is a rare, highly aggressive tumour, which accounts for less than 1% of all cancer deaths (1). This neoplasm arises from the surface serosal cells of the pleural (>90% of cases), peritoneal, and pericardial cavities and from the tunica vaginalis of the testis (2). Although the association between exposure to asbestos and the development of mesothelioma is commonly accepted, the exact mechanism as a result of which asbestos induces the mesothelioma is unknown (3). The prognosis is generally poor with a reported median survival of 4-12 months in either untreated or treated (surgery, radiotherapy, or chemotherapy) patients (4). Moreover, mesothelioma has proved resistant to classical chemotherapeutic and radiation regimes and the natural history of the disease has not been influenced by standard therapy (5-8). Several studies report the importance of cell cycle proteins in the pathogenesis and the prognosis of this neoplasm (9). A precise regulation of the cell cycle is a pre-requisite for the homeostasis of the eukaryotic cell. Over the last decade, scientists successfully delved into the molecular machinery devoted to the fine regulation of the cell cycle phases, identifying and characterizing several genes and gene products involved (10). A key role is played by cell cycle kinases (cdk), relatively small proteins with an apparent molecular mass between 33 and 43 kda, whose activity is regulated by the arrangement in a multimeric complex with larger proteins, called "cyclins", after their cyclical expression and degradation during the cell cycle. Different cdk/cyclin complexes, formed with clear-cut timing throughout the cell cycle, together with their phosphorylation/dephosphorylation, efficiently regulate the activity of the multimeric holoenzyme. Conversely, cdk/cyclin complexes are negatively modulated by the binding of a family of small proteins called cdk inhibitors; namely the CIP (p21 and p27) and the INK (p16, p15, p19 and p18) families (11,12). The p53 tumour suppressor gene is also involved in cell-cycle checkpoints by virtue of its action as a transcription factor for several cell-cycle regulatory proteins, including the p21 gene (13). On the other hand, proliferating cell nuclear antigen (PCNA) is involved in activation of DNA polymerase, a function required for DNA replication and repair (14,15). Finally, the p53 to p21 pathway also inhibits DNA replication by merit of p21's interaction with PCNA, without affecting PCNA's DNA repair abilities (16,17). The retinoblastoma gene family consists of three members, the product of the retinoblastoma gene (prb), which is one of the most studied tumour suppressor genes, and two related proteins, prb2/p130 and p107, which have been shown to be structurally and functionally similar to prb (18). Sequence analysis of these two proteins shows they share large regions of homology with prb, especially in two discontinuous domains which make up the "pocket region" (19,20). The pocket domain is required for binding the three members of the Rb-family with several viral transforming oncoproteins, as well as with members of the E2F family (21). Both prb2/p130 and p107, like prb, display growth suppressive properties, although the 443

E. P. Spugnini et al. growth arrest mediated by the three pocket proteins is not identical. This suggests that, although the different members of the retinoblastoma gene family may complement each other, they are not fully redundant functionally (22,23). The Rb pocket proteins (prb, p107 and p130) play a critical role in G1/S progression, at least in part, through binding and inactivation of factors (eg, E2F) that promote transcription of genes required for DNA replication (24). Although prb2/p130, p107, similarly to prb, interact with members of the E2F transcription family and have similar functional consequences, each pocket protein has a different temporal profile of interaction with different E2F/DP1 complexes. The binding of prb2/p130 to these complexes is detected predominantly during G0, while that of p107 is detected during the G1 and S phases (25-28). In figure 1 the most important cell cycle molecules involved in cancer pathogenesis and progression are illustrated. Cell Cycle genes in mesothelioma As far as mesothelioma is concerned, most of the studies about cell cycle regulation in this neoplasm have been performed on the G1/S phase. First studies about cell cycle protein interference in mesothelioma date lack to 1998. In that period Beer et al. (29) investigated the expression of the Ki-67 nuclear antigen detected by MIB-1 immunoreactivity as an indicator of cell proliferation and survival in pleural malignant mesotheliomas. Ki-67 is a well known nuclear antigen highly expressed in proliferating cells, routinely used as a prognostic factor in the assessment of tumour behaviour (30). Beer and coll. showed that patients with a MIB-1 proliferation index less than 30% had a significantly longer survival than patients with a proliferation index exceeding that value. Similar observations were confirmed by Comin et al. in 2000 (31). A low mitotic count and a low apoptotic index defined by in situ end-labelling were also found to be associated with a significant survival advantage. The proliferation index, the mitotic count, and the apoptotic index, therefore, were proposed as useful prognostic markers in pleural malignant mesotheliomas (32). In 2001 Bongiovanni and colleagues (33) provided the first evidence of a possible role of p27kip1 in predicting survival in a large series of long-term survival patients. As described previously, p27kip1 acts as inhibitor during the cell cycle progression from the G1-phase to the S-phase; therefore, this protein is defined a cell cycle regulator and a putative tumour suppressor gene (34,35). In this paper the authors described as p27kip1 positively correlates to the dura- Fig. 1 - Schematic representation of the cell cycle clock with the molecules involved in its regulation. 444

Cell-Cycle Molecules in Mesothelioma tion of survival: all patients with survival times exceeding 24 months had high p27kip1 values. They also found that the MIB-1/Ki-67 positivity, instead, was inversely correlated to the survival. In the same work it was also reported that the combined evaluation of Ki-67 and p27kip1 led to additional reliability in the prediction of survival with a 100% specificity and sensitivity. Nuclear staining intensity of p27kip1 was found to be associated with survival: the higher the intensity, the longer the survival. On the other hand, the cytoplasmic localization of p27kip1 immunoreactivity was of little significance. Gunther et al., however, showed such a localization to be a common event in rectal carcinoma and proved it to be the best prognostic factor of disease-free survival in such neoplasms (36). Moreover, in this study patients with elevated cytoplasmic p27kip1 expression showed a significantly poorer outcome, irrespective of their nuclear staining. Further investigations are needed to explain whether the nuclear and cytoplasmic distribution reflect two different biologic activities or two different regulatory mechanisms of p27kip1. Histologic subtyping was considered one of the more useful criteria to recognize lesions with a favourable prognosis: the epithelioid type is usually considered less aggressive than the biphasic and pure sarcomatoid types. However, the exact identification of the subtype could be difficult on small biopsy specimens especially in the presence of mixed lesions. Johansson and colleagues showed that p27kip1 immunoreactivity was significantly higher in pure epithelioids compared to biphasic ones and they found also that Ki-67 had opposite expression in the two histotypes. This observation could provide a possible biologic counterpart to the usual, although not univocally recognized, more favourable prognosis of epithelioid mesothelioma (37). Several studies have investigated the potential prognostic value of other Cip/Kip inhibitors, as p53 and p21, and the role of the SV40 large T antigen (L- Tag) in malignant mesotheliomas. It has been proposed that malignant transformation of human mesothelioma cells does not require inactivation of p53 by point mutations in the conserved regions of the gene (38). Moreover, it has been demonstrated that p53 over-expression does not correlate in a statistically significant manner with survival, but its over-expression is a frequent feature of pleural mesothelioma and proves useful for routine differentiating between malignant and non-neoplastic mesothelial alterations (39). In 2001 Yang et al.. (40) proposed that p53 is inactive in malignant pleural mesothelioma cells as a result of p14 ARF deficiency. Homozygous deletions of INK4a/ARF locus have been shown to be the predominant events which occur at a frequency of >70% in this malignancy (41,42). The INK4a/ARF locus (43) on human chromosome 9p21 plays an important role in both the prb and p53 tumour suppressor pathways by encoding two distinct proteins translated from alternatively spliced mrnas: p16 INK4a regulates prb phosphorylation and induces cell cycle arrest in G1 phase (44); p14 ARF plays a role as a negative regulator of MDM2, interfering with MDM2-mediated shuttling and degradation of p53 (45,47). A single mutational event at the INK4a/ARF locus therefore has the potential to disrupt both prb and p53 tumour suppressor pathways. In a recent study, Hopkins-Donaldson et al. (48) investigated the status of p53 in malignant pleural mesothelioma cells lacking p14 ARF. In contrast with the previous observations, their findings suggest that p53 is functional in malignant pleural mesothelioma in the absence of p14 ARF ; it has been found that p53 is activated by DNA damage, and in this way it contributes to the apoptotic response induced by cisplatin (CDDP). Moreover, Hopkins-Donaldson and colleagues also demonstrates that removal of downstream inhibitors such as survivin can enhance this response more successfully than the disruption of p53-mdm2 interaction by p14 ARF. As for to the role of the SV40 protein, De Luca and colleagues reported SV40-like sequences in 86% of 35 archival specimens of mesothelioma. They demonstrated that SV40 Tag, isolated from frozen biopsies of human mesothelioma, binds each of the retinoblastoma family proteins, prb, p107 and prb2/p130. It was assumed that the tumorigenic potential of SV40 Tag in some human mesotheliomas may arise from its ability to interact with and thereby inactivate several tumour and/or growth suppressive proteins (49). Baldi et al. have investigated the expression of p21 in a group of mesothelioma specimens already characterised for the expression of p53 and for the presence of SV40 sequences (50). In this study they did not found any relationship between p21 expression and histological type in the mesothelioma specimens, but a significant positive relationship between p21 expression level and the overall survival of the patients was found. Thence, the p21 pathway is involved in the pathogenesis of mesothelioma. This could occur by silencing p21 through p53 inactivation by SV40 proteins and/or the weaker activation of p21 by alternative pathways. A greater reduction in p21 expression will 445

E. P. Spugnini et al. cause greater aggression and result in a poorer prognosis. In a different work, Baldi et al. analyzed the potential prognostic value of the immunohistochemical expression of cyclooxygenase-2 (COX-2) and p27 in 29 malignant mesotheliomas already screened for the expression of p21 and p53 (51). COX-2 has been implicated in the carcinogenesis of several neoplasms and its over-expression has been noted in many solid tumours and has been correlated with a worse prognosis in colorectal cancer, non-small cell lung cancer, and gastric cancer (52). A recent study showed that COX-2 expression is a strong prognostic factor in human mesothelioma, which independently contributes to the other clinical and histopathological factors in determining a short survival (53). It has been also proposed that COX-2 exerts its influence on mesangial cell proliferation in vitro by a novel mechanism involving the tumour suppressor p53 and the cell cycle inhibitors p21 and p27 (54). In this work Baldi et al. did not find any relationship between the levels of expression of the COX-2, p27, p21 and p53 and their patients histological types, but they observed that a high level of COX-2 expression and low p21 and p27 expression were associated with a statistically significant decrease in survival. These data agree with the lack of correlation reported by others between COX-2, p21 and p53 and type of mesothelioma (55,56). Other groups have found a significant correlation between p27 expression and epithelioid histotype (57,58). The data suggest that COX-2 expression may be a useful prognostic parameter and thus support further investigations into the clinical usefulness of COX-2 inhibitors in the treatment of malignant mesotheliomas. In a recent study by Vivo et al. (59) the anti-tumoral activity of recombinant human interferon gamma (rhu-ifnγ) on nine different human malignant mesothelioma cell lines has been investigated. The authors showed that r-hu-ifngamma produced an arrest in the G1 and G2-M phases of the cell cycle, associated with a reduction in both cyclin A and cyclin dependent kinase inhibitors (CDKIs) expression. Moreover, cyclin B1/cdc2 activity was decreased. The cell cycle arrest induced by r-hu-ifngamma seems to depend on cyclin regulation through p21 (WAF1/CIP1)- and p27(kip1) - independent mechanisms and it is not directly related to the induced DNA damage. Another interesting study by Mukohara et al. (60) investigated a potential therapeutic role for the Met receptor in malignant pleural mesothelioma. Expression of the Met receptor and its ligand, hepatocyte growth factor (HGF), has been observed in 74% to 100% and 40% to 85% of malignant pleural mesothelioma specimens (61,62). HGF stimulation has been shown to enhance malignant pleural mesothelioma cell proliferation, migration, cell scattering, and invasiveness (63). The authors provided evidence supporting the possibility of therapeutic targeting HGF/Met signalling in malignant pleural mesothelioma. Disrupting HGF/Met signalling is potentially attractive as it may not only inhibit malignant pleural mesothelioma growth but also alter the rate of migration and invasion in a disease that is clinically characterized by local extension. However, the effects of Met receptor inhibition are limited to a minority of malignant pleural mesothelioma cell lines, those that also produce HGF. This study would predict that clinical trials of Met inhibitors, such as PHA-665752, in malignant pleural mesothelioma would be most effective in patients whose tumours express both Met and HGF. Finally, a recent work by Adusumilli and colleagues analyzed the effects of a potential oncolytic viral therapy in the treatment of malignant pleural mesothelioma (64). The authors showed that cisplatin induces GADD34 (Growth Arrest and DNA Damage-Inducible Protein) expression in ten human malignant pleural mesothelioma cell lines, and that this up-regulation selectively enhanced the cytotoxicity of the gamma134.5-deficient oncolytic virus, NV1066. This provides a cellular basis for combination therapy with cisplatin and NV1066 to treat malignant pleural mesothelioma and achieve synergistic efficacy, while minimizing dosage and toxicity. Conclusion All the works mentioned in this review would provide useful information on the prognosis of newly diagnosed cases and would allow researchers to recognize a subgroup of patients with significantly improved survival, in which it could be possible to achieve better response to therapy. Nevertheless, targeting multiple checkpoint proteins may represent a good therapeutic strategy for the development of new molecular treatments for pleural mesothelioma (65). The data presented in this article support this hypothesis and strongly suggest further works aimed at investigating the simultaneous expression of numerous cell cycle regulators in mesotheliomas. Acknowledgements. This work has been supported by "Grant 2006" of the Italian Ministry of Health and FIRB/MUR to E.P.S. and G.C., and by grants from 446

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