Cell Cycle Tumor suppressor activity of CBX7 in lung ...

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Tumor suppressor activity of CBX7 in lung carcinogenesis a

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Floriana Forzati , Antonella Federico , Pierlorenzo Pallante , Monica Fedele & Alfredo a

Fusco a

Istituto di Endocrinologia ed Oncologia Sperimentale del CNR; Dipartimento di Biologia e Patologia Cellulare e Molecolare; Facoltà di Medicina e Chirurgia di Napoli; Università degli Studi di Napoli “Federico II”; Naples, Italy Published online: 15 May 2012.

To cite this article: Floriana Forzati, Antonella Federico, Pierlorenzo Pallante, Monica Fedele & Alfredo Fusco (2012) Tumor suppressor activity of CBX7 in lung carcinogenesis, Cell Cycle, 11:10, 1888-1891, DOI: 10.4161/cc.20022 To link to this article: http://dx.doi.org/10.4161/cc.20022

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Cell Cycle 11:10, 1888-1891; May 15, 2012; © 2012 Landes Bioscience

Tumor suppressor activity of CBX7 in lung carcinogenesis Floriana Forzati, Antonella Federico, Pierlorenzo Pallante, Monica Fedele and Alfredo Fusco* Istituto di Endocrinologia ed Oncologia Sperimentale del CNR; Dipartimento di Biologia e Patologia Cellulare e Molecolare; Facoltà di Medicina e Chirurgia di Napoli; Università degli Studi di Napoli “Federico II”; Naples, Italy

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he generation of knockout mice for the Cbx7 gene validates the tumor suppressor role of CBX7, whose expression is lost in several human malignancies. Indeed, these mice developed liver and lung adenomas and carcinomas. Cyclin E overexpression due to the lack of Cbx7-negative regulation of its expression likely accounts for the phenotype of the Cbx7-KO mice. A similar mechanism is likely involved in human lung carcinogenesis, since Cyclin E upregulation associated with the loss of CBX7 expression has been observed in most of the human lung carcinomas analyzed.

silencing of PcG target genes is frequent.5 Indeed, EZH2 is amplified and highly expressed in many tumor types, and its expression correlates with a high proliferation rate and a poor outcome in breast, prostate and other cancers.6,7 Conversely, the CBX7 gene is drastically downregulated in thyroid carcinomas, and its expression progressively decreases with malignant grade and neoplastic stage.8 Moreover, further studies have shown that the correlation of the loss of CBX7 with a highly malignant phenotype and a consequent poor prognosis is a general event in oncology. In fact, the loss of CBX7 expression has been recently shown to be associated with increasing malignancy grade in colon,9 bladder,10 pancreatic,11 breast,12 gastric13 and lung carcinoma,14 whereas the retention of CBX7 expression correlates with a longer survival.9 However, the role of CBX7 in the regulation of cell growth and tumorigenesis is still controversial, since there is evidence that its overexpression would lead to cellular immortalization in vitro and tumor development in vivo, thereby proposing CBX7 as an oncogene.2,15 To address this issue, we generated and characterized mice null for the Cbx7 gene. In our paper by Forzati et al. 2012 we report that the Cbx7-KO mice develop liver and lung adenomas and carcinomas. Interestingly, the development of benign or malignant lesions is quite well correlated with the number of Cbx7 functioning alleles. In fact, both liver and lung carcinomas mostly develop in the mice where both the Cbx7 alleles are impaired.14 Just recently, we have better characterized the early histological lung anomalies: at the third month of age, almost all the Cbx7-/- mice

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Key words: CBX7, Wng tumor, KO mice, cyclin E, HMGA1 Submitted: 02/21/12 Accepted: 03/13/12 http://dx.doi.org/10.4161/cc.20022 *Correspondence to: Alfredo Fusco; Email: [email protected]

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Polycomb group proteins function as multiprotein complexes and are part of a gene regulatory mechanism that determines cell fate during normal and pathogenic development. They form transcriptional repressor modules that can be functionally divided into at least two distinct complexes: (1) the initiation complex, polycomb repression complex 2 (PRC2), the core of which in humans consists of EZH2, EED and SUZ12; (2) the maintenance complex PRC1, whose components are the mammalian homologs of Drosophila polycomb (Pc), posterior sex combs (Psc), sex combs extra (Sce) and polyhomeiotic (Ph).1 CBX7 is a polycomb homolog consisting of a conserved chromodomain near the N terminus and a polycomb box in the carboxyterminal region.2 Mouse Cbx7 associates with facultative heterochromatin and with the inactive X chromosome, suggesting a role of the Cbx7 protein in the repression of gene transcription.3,4 Deregulation of PcG proteins contributes to tumorigenesis, where aberrant

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show lung atelectasia that is clearly due to the obstruction of the bronchioli caused by hyperproliferation of the surface epithelial cells. Moreover, clear signs of dysplasia are also present at this time (Forzati F, et al. unpublished observations). Therefore, these results, besides proposing the Cbx7-KO mice as an excellent model to study lung cancer progression, clearly indicate a tumor suppressor role of CBX7 in carcinogenesis. This conclusion is also supported by further evidences: (1) It has been shown that the restoration of CBX7 expression in thyroid, gastric and colon carcinoma cell lines inhibits cell growth with an accumulation of the cell population in the G1 phase of the cell cycle, suggesting a negative role of CBX7 in the control of the G1/S transition of the cell cycle. Consistently, the Cbx7-KO MEFs grow faster than the wild-type controls, with accumulation of the cell population in the S phase of the cell cycle, whereas the MEFs deriving from transgenic mice overexpressing Cbx7 have a reduced growth rate with respect to the wild-type MEFs and accumulate in G0 /G1. (2) It has been recently demonstrated that CBX7 is able to positively regulate the expression of the gene coding for E-cadherin,16 which is known to play a critical role in maintaining normal epithelial cell morphology, and whose loss of expression represents a general feature of the epithelial-mesenchymal transition (EMT).17,18 In fact, it has been shown that CBX7 is able to preserve the expression of E-cadherin by interacting with histone deacetylase 2 and inhibiting its activity on the CDHI promoter.16 Consistently, a direct correlation between the levels of E-cadherin and CBX7 expression has been reported in thyroid and pancreatic carcinomas.9,16 Moreover, CBX7 is also able to negatively regulate the expression of other important genes involved in EMT. Indeed, preliminary data produced in our laboratory show that CBX7 is able to repress the expression of S100A4 and osteopontin, two genes essential for the acquisition of the fully malignant phenotype.19,20 In our study, we have also attempted to unveil the mechanism by which the loss of the CBX7 expression might induce lung tumors. We have concentrated our attention on the CCNE1 gene, coding

for Cyclin E, because: (1) previous data showed overexpression of this gene in human lung carcinoma;21 (2) transgenic mice overexpressing Ccne1 developed dysplasia and multiple pulmonary adenocarcinomas;22 (3) Cyclin E has a key role in the control of the G1/S transition;23 (4) Cyclin E overexpression induces chromosomal instability.24 We have reported that CBX7 protein is able to bind the CCNE1 promoter, where it recruits histone deacetylase 2 (HDAC2) and negatively regulates Cyclin E expression. Moreover, we show that CBX7 and HMGA1 proteins interact on this promoter acting in an opposite way: one protein, CBX7, inhibiting, the other one, HMGA1, activating the CCNE1 promoter activity. A scheme of the CBX7HMGA1 competition on the CCNE1 promoter is shown in Figure 1. Therefore, the loss of the CBX7 expression would lead to increased CCNE1 promoter activity prevailing the activating function of HMGA1. Consistently, Cyclin E overexpression was observed in Cbx7-KO tissues and MEFs with respect to the wild-type counterparts. A relevant result of the paper by Forzati et al. is that upregulation of Cyclin E due to the lack of the negative control exerted by CBX7 seems to have a critical role also in human lung carcinogenesis, since Cyclin E overexpression associated with the loss of CBX7 expression has been observed in almost all the human lung carcinomas analyzed. Interestingly, a clear decrease in CBX7 expression associated with Cyclin E overexpression and LOH at the CBX7 locus has been observed also in the “morphologically normal” tissue adjacent to lung tumor, proposing, then, the downregulation of CBX7 expression as a critical step in human lung cancer progression. Consistently, the restoration of CBX7 expression in two lung carcinoma cell lines resulted in an inhibitory effect on cell growth, leading to an increased Cyclin E expression. However, even though the increased expression of Cyclin E likely accounts for the neoplastic phenotype of the Cbx7-KO mice, other experiments are required to validate this hypothesis. The generation of mice carrying the disruption of both Cbx7 and Ccne1 genes is needed; the lack of development of lung carcinomas

in these mice would validate our hypothesis. Moreover, it is reasonable to retain that the loss of CBX7 contributes to carcinogenesis also by the regulation of other genes, as described above. Preliminary results indicate CCNA1, encoding the Cyclin A, as a gene repressed by CBX7, and previous results of our group also suggest that miRNA deregulation, consequent to the lack of CBX7 expression, might be involved in the cancer progression. Indeed, we have previously shown that CBX7 negatively controls expression of miR-181b, which, in turn, targets CBX7.12 Since CBX7 is downregulated by HMGA1, which, in turn, is able to upregulate miR-181b, we can retain that CBX7 is involved in a pathway, also including miR-181b and HMGA, that may give an important contribution to the progression step of carcinogenesis. Another open question is to verify how the lack of the CBX7 expression may influence the lung cancer progression induced by Ki-ras mutations that are the most frequent genetic lesion found in lung neoplasias.25 To this aim, we will cross the Cbx7-KO mice with transgenic mice expressing a mutated Ki-ras gene that develop a broad spectrum of multifocal lesions in lungs, ranging from bronchioloalveolar hyperplasias to large bronchioloalveolar adenomas and adenocarcinomas.26 It has been previously reported that CBX7 extends the lifespan of a wide range of normal human cells and immortalizes mouse fibroblasts by downregulating expression of the Ink4a/Arf locus.2,27 These results would indicate CBX7 as an oncogene, whereas the phenotype of the Cbx7-KO mice proposes CBX7 as a tumor suppressor. It is conceivable that CBX7 can exert both oncogenic and anti-oncogenic functions depending on the nature of other cellular events and on the presence of interacting proteins, which depends on the cellular context. This is the case of another PcG gene, namely EZH2. Indeed, this gene is overexpressed and acts as an oncogene in several human malignancies.6,7 However, very recently, somatic mutations of the EZH2 gene, located within an exon encoding the catalytic SET domain of the protein, have been detected in B-cell lymphomas of germinal-center origin. These mutations


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Figure 1. Competition between HMGA1 and CBX7 for regulation of the CCNE1 promoter. This model suggests a mutual competition between CBX7 and HMGA1 for the binding to the CCNE1 promoter: in Cbx7+/+ mice, CBX7 negatively regulates CCNE1 expression by recruiting HDAC2 on the CCNE1 promoter and avoiding that HMGA1 binds to it; in Cbx7+/- mice, the reduced levels of CBX7 allow HMGA1 to displace CBX7 from the CCNE1 promoter, enhancing transcription; in Cbx7-/- mice, HMGA1, without any competition from CBX7, strongly activates the CCNE1 promoter.

result in a decrease of the histone methyltransferase activity of the EZH2 protein,28 leading to its loss of function, which suggests that EZH2 is a tumor suppressor gene. Interestingly, the generation of Cbx7-KO mice has also evidenced a role of the Cbx7 gene in the control of physiological functions, such as adipogenesis. Indeed, the analysis of the heterozygous and homozygous Cbx7-KO mice showed a significant increase in fat tissue. Then, further studies have confirmed the role of Cbx7 in adipogenesis. In fact, a decreased Cbx7 expression was observed when the pre-adipocytic cells 3T3L1 were induced toward the adipocyte differentiation. Moreover, MEFs and ES cells in which the Cbx7 gene is disrupted differentiate to adipocyte much more efficiently than the respective wild-type cells (Forzati et al. manuscript in preparation). In conclusion, our recent paper reporting the generation of mice knockout for the Cbx7 gene finally validates a tumor

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suppressor role for this gene and confirms the role of the loss of CBX7 expression in the development of highly aggressive neoplasias. Acknowledgments

This work was supported by grants from AIRC (IG 5346) and the Ministero dell’Università e della Ricerca Scientifica e Tecnologica—MIUR (PRIN 2008). References 1. Valk-Lingbeek ME, Bruggeman SW, van Lohuizen M. Stem cells and cancer; the polycomb connection. Cell 2004; 118:409-18; PMID:15315754; http:// dx.doi.org/10.1016/j.cell.2004.08.005. 2. Gil J, Bernard D, Martínez D, Beach D. Polycomb CBX7 has a unifying role in cellular lifespan. Nat Cell Biol 2004; 6:67-72; PMID:14647293; http:// dx.doi.org/10.1038/ncb1077. 3. Bernstein E, Duncan EM, Masui O, Gil J, Heard E, Allis CD. Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin. Mol Cell Biol 2006; 26:2560-9; PMID:16537902; http:// dx.doi.org/10.1128/MCB.26.7.2560-9.2006. 4. Lund AH, van Lohuizen M. Polycomb complexes and silencing mechanisms. Curr Opin Cell Biol 2004; 16:239-46; PMID:15145347; http://dx.doi. org/10.1016/j.ceb.2004.03.010.

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11. Karamitopoulou E, Pallante P, Zlobec I, Tornillo L, Carafa V, Schaffner T, et al. Loss of the CBX7 protein expression correlates with a more aggressive phenotype in pancreatic cancer. Eur J Cancer 2010; 46:1438-44; PMID:20185297; http://dx.doi. org/10.1016/j.ejca.2010.01.033. 12. Mansueto G, Forzati F, Ferraro A, Pallante P, Bianco M, Esposito F, et al. Identification of a new pathway for tumor progression: microRNA-181b upregulation and CBX7 downregulation by HMGA1 protein. Genes Cancer 2010; 1:210-24; PMID:21779448; http://dx.doi.org/10.1177/1947601910366860. 13. Jiang Z, Guo J, Xiao B, Miao Y, Huang R, Li D, et al. Increased expression of miR-421 in human gastric carcinoma and its clinical association. J Gastroenterol 2010; 45:17-23; PMID:19802518; http://dx.doi. org/10.1007/s00535-009-0135-6. 14. Forzati F, Federico A, Pallante P, Abbate A, Esposito F, Malapelle U, et al. CBX7 is a tumor suppressor in mice and humans. J Clin Invest 2012; 122:61223; PMID:22214847; http://dx.doi.org/10.1172/ JCI58620. 15. Scott CL, Gil J, Hernando E, Teruya-Feldstein J, Narita M, Martínez D, et al. Role of the chromobox protein CBX7 in lymphomagenesis. Proc Natl Acad Sci USA 2007; 104:5389-94; PMID:17374722; http://dx.doi.org/10.1073/pnas.0608721104. 16. Federico A, Pallante P, Bianco M, Ferraro A, Esposito F, Monti M, et al. Chromobox protein homologue 7 protein, with decreased expression in human carcinomas, positively regulates E-cadherin expression by interacting with the histone deacetylase 2 protein. Cancer Res 2009; 69:7079-87; PMID:19706751; http://dx.doi.org/10.1158/0008-5472.CA N-091542.

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