Genetic and epigenetic alterations of RB2/p130 tumor suppressor gene in human sporadic retinoblastoma: implications for pathogenesis and therapeutic approach Gian Marco Tosi 1 , Carmela Trimarchi 2 , Marcella Macaluso 3 , Dario La Sala 4,5 , Alfredo Ciccodicola 6 , Stefano Lazzi 4 , Mina Massaro-Giordano 7 , Aldo Caporossi 1 , Antonio Giordano 3,4 and Caterina Cinti* ,3,5 1 Department of Ophthalmology and Neurosurgery, University of Siena, Siena, Italy; 2 Institute of Neuroscience CNR, Pisa, Italy; 3 Sbarro Institute of Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA, USA; 4 Department of Human Pathology and Oncology, University of Siena, Siena, Italy; 5 Institute of Clinical Physiology CNR, Siena, Italy; 6 Institute of Genetics and Biophysics CNR, Napoli, Italy; 7 Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA Human retinoblastoma occurs in two forms (familial and sporadic) both due to biallelic mutation of the RB1/p105 gene even if its loss is insufficient for malignancy. We have recently reported that loss of expression of the retino- blastoma-related protein pRb2/p130 correlates with low apoptotic index, suggesting that RB2/p130 gene could be involved in retinoblastoma. Mutational analysis of RB2/ p130 in primary tumors showed a tight correlation between Exon 1 mutations and pRb2/p130 expression level in sporadic retinoblastoma. These mutations are located within a CpG-enriched region prone to de novo methylation. Analysis of RB2/p130 methylation status revealed that epigenetic events, most probably consequent to the Exon 1 mutations, determined the observed phenotype. Treatment of Weri-Rb1 cell line by 5-Aza- dC induced an increase in expression level of pRb2/p130, E2F1, p73 and p53. Overall, our results highlight a crucial role of epigenetic events in sporadic retinoblastoma, which opens a perspective for new therapeutic approaches. Oncogene (2005) 24, 5827–5836. doi:10.1038/sj.onc.1208630; published online 20 June 2005 Keywords: sporadic retinoblastoma; Rb2/p130 mutation; methylation; demethylating agent; 5-Aza-dC Introduction On the path to becoming cancerous, normal mammalian cells evolve to an atypical organization. Normal tissues and cells are the product of a complex set of genes expressed at different levels, which respond to internal and external stimuli to alter level of expression. Cancer cells do not express the same set of genes as their normal counterparts, and such anarchy is a hallmark of the cancerous state (Hanahan and Weinberg, 2000). The change in gene expression profiles evident in cancer cells depends on alterations at the genetic (i.e. mutations) as well as epigenetic (i.e. transcriptional inactivation due to methylation) level (Issa, 1999; Knudson, 2002). Retino- blastoma is the most common intraocular malignancy in children. Human retinoblastoma occurs in two forms: a nonheritable form, which is usually unilateral, and a heritable form, which is often bilateral with autosomal dominant expression. Both forms have been ascribed to biallelic mutation of the Rb1/p105 gene and the consequent loss of its tumor-suppressive functions. In the familial form, one mutated copy is inherited from an affected parent and a further mutation accumulates to determine the disease, while in the sporadic form both mutations occur spontaneously (Knudson et al., 1975). The basic function of pRb1/p105 is to hold cells in G1 or G0 phase of the cell cycle and prevent entry into S phase by interacting and negatively regulating the E2F family of transcription factors. Moreover, pRb1/p105 is also involved in the apoptotic response by interacting with p53 pro-apoptotic pathway (Hsieh et al., 1997, 1999; Chan et al., 2001). Not withstanding the fact that mutation of Rb1/p105 is common to all retinoblastomas, much evidence indicates that loss of pRb1/p105 from a developing retinal cell is insufficient for malignancy (DiCiommo et al., 2000). The possibility that further mutations of other tumor suppressor genes could occur in sporadic retinoblastoma is supported by recent studies providing direct evidence that loss of pRb1/ p105 function leads to genome instability and predispose to cancer by increasing DNA mutation rate (Zheng and Lee, 2002). On the other hand, in tumorigenesis, genomic instability is tightly linked to deregulation of DNA methylation pattern and partial or total gene silencing may be achieved by this epigenetic mechanism (Sugimura and Ushijima, 2000). This is the case also for retinoblastoma where hypermethylation of both RB1/p105 and other genes has been shown (Choy et al., 2004). pRb1/p105 functions are shared by two Received 15 December 2004; revised 8 February 2005; accepted 9 February 2005; published online 20 June 2005 *Correspondence: C Cinti, Institute of Clinical Physiology, IFC-CNR, Siena, c/o Department of Human Pathology and Oncology, Patholo- gical Anatomy Unit, via delle Scotte 6, 53100 Siena, Italy; E-mail: cinti@area.bo.cnr.it Oncogene (2005) 24, 5827–5836 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc