Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression

Background: Cervical Cancer ( CC) exhibits highly complex genomic alterations. These include hemizygous deletions at 4p15.3, 10q24, 5q35, 3p12.3, and 11q24, the chromosomal sites of Slit-Robo pathway genes. However, no candidate tumor suppressor genes at these regions have been identified so far. Slit family of secreted proteins modulates chemokine-induced cell migration of distinct somatic cell types. Slit genes mediate their effect by binding to its receptor Roundabout ( Robo). These genes have shown to be inactivated by promoter hypermethylation in a number of human cancers. Results: To test whether Slit-Robo pathway genes are targets of inactivation at these sites of deletion, we examined promoter hypermethylation of SLIT1, SLIT2, SLIT3, ROBO1, and ROBO3 genes in invasive CC and its precursor lesions. We identified a high frequency of promoter hypermethylation in all the Slit- Robo genes resulting in down regulated gene expression in invasive CC, but the inhibitors of DNA methylation and histone deacetylases (HDACs) in CC cell lines failed to effectively reactivate the down-regulated expression. These results suggest a complex mechanism of inactivation in the Slit- Robo pathway in CC. By analysis of cervical precancerous lesions, we further show that promoter hypermethylation of Slit- Robo pathway occurs early in tumor progression. Conclusion: Taken together, these findings suggest that epigenetic alterations of Slit- Robo pathway genes (i) play a role in CC development, (ii) further delineation of molecular basis of promoter methylation-mediated gene regulation provides a potential basis for epigenetic-based therapy in advanced stage CC, and (iii) form epigenetic signatures to identify precancerous lesions at risk to progression.