DNA methylation changes are involved in both the initiation of carcinogenesis and progression. We have demonstrated genome-scale alterations in DNA methylation patterns in urothelial tumors and also normal-appearing tissues in bladders with cancer. We also found that hypomethylation of non-CpG island regions seems to be preferentially associated with low stage Ta-T1 disease providing additional molecular evidence that there are two distinct molecular pathways to carcinoma of the bladder (4
). Interest in the field of DNA methylation and cancer was originally spurred by early observations that tumors were often hypomethylated relative to normal tissues (34
), the majority of which was assumed to occur at repetitive elements. It has been suggested that such hypomethylation could lead to genomic instability and contribute to the tumor phenotype (35
A systematic analysis of DNA methylation of various types of repetitive elements in urothelial tumors revealed that both Sat-α and LINE-1 elements are significantly hypomethylated (37
). We have recently revealed one consequence of hypomethylation of LINE-1 elements in UC is the aberrant activation of transcripts of nearby genes (38
). However, while LINE-1 promoters contain a small CpG island, the relationship between DNA methylation of non-CpG islands and expression remains controversial and underexplored even though approximately 45% of all human genes have promoters that are not located within CpG islands (39
). DNA hypomethylation associated with increased gene expression has been reported in several studies (40
). We have recently confirmed a correlation between increased expression and hypomethylation at non-CpG island promoters in prostate cancer cell lines (32
). In addition, it is possible that lower levels of DNA methylation may be related to a less malignant phenotype. For instance, when expression of the DNA methyltransferase Dnmt1 is reduced in ApcMin/+
mice, fewer intestinal tumors occur but more early intestinal lesions form, supporting opposing effects of DNA hypomethylation initiating lesions but suppressing further progression (42
One of the most clinically relevant questions in tumor biology is why some tumors grow and progress while others remain quiescent (43
). The high incidence of in situ
tumors discovered during autopsies indicates that there is a rarely occurring additional step required for a tumor to become life threatening (43
). Such a step has been postulated to include an angiogenic switch (43
) or alterations in the stroma (44
). Our results suggest that a switch to accumulating DNA hypermethylation may be involved, such as could be caused by overexpression of DNA methyltransferases (45
We have also revealed that the urothelium in bladders with cancer is no longer “normal”. Instead, the urothelium in these diseased bladders has undergone widespread epigenetic alterations mainly consisting of aberrant hypermethylation. Our findings support a field-cancerization model where independent events occur across the urothelium resulting in a field defect that is polyclonal (). The presence of an epithelial-wide defect in bladders with tumors is quite astounding. In contrast to work in other organs such as colon (18
), esophagus (17
), breast (19
), and stomach (20
), we found no dependency on the distance from the tumor. This significant difference in the pathogenesis of UC is also supported by the unique characteristics of tumor multifocality and a tendency to recur. The most likely underlying cause is that the urothelium is uniformly exposed to carcinogenic compounds excreted in the urine, and once a tumor forms, it can be assumed that the entire urothelium has been exposed to critical levels of carcinogens (45
). It is also possible that the presence of the tumor itself leads to epigenetic alterations across the bladder. We observed that 83% of the hypermethylated loci found in the corresponding urothelium were also found in invasive tumors. Given such a high degree of similarity between the two tissues, we believe the most likely explanation is that these tissues are at two different steps in the tumorigenic pathway.
While the alterations in DNA methylation that occur across the entire urothelium do not directly confer a growth advantage since polyclonality of the urothelium is maintained, they may still have functional significance. The role of the tight junction binding protein ZO2 in cancer has not been studied in depth, but expression of other components of tight junctions is frequently altered in cancer, with loss of expression leading to increased cell motility and invasiveness. Increased DNA methylation of MYOD1
has previously been shown to be associated with oncogenic transformation (46
has previous been found to be hypermethylated in premalignant gastric tissue (47
) and loss of expression is associated with increased invasion in melanocytes (48
). We identified a total of 72 genes as hypermethylated in corresponding normal-appearing tissues and associated with down-regulation in tumors. Taken together, the DNA methylation at these and other loci in apparently normal urothelium may contribute to a loss of epithelial integrity across the entire bladder. Such an epithelial-wide defect could allow for a more permissible environment for the growth of newly mutated cells.
One limitation of our study is that we are unable to determine whether the widespread field defect identified in bladders with invasive tumors is also present in bladders with non-invasive tumors. All of the corresponding normal-appearing tissues that we collected were from patients that were having their entire bladders removed. Treatment of non-invasive urothelials tumors usually involves surgical excision of the tumor and does not result in the collection of additional normal-appearing tissue. Therefore, since we have found that non-invasive and invasive urothelial tumors appear to undergo two separate epigenetic pathways to tumorigenesis we cannot rule out that the generalized epigenetic defect we have uncovered is specific to bladders with invasive tumors.
In summary, urothelial tumors develop along two separate molecular pathways that differ both genetically and epigenetically. A generalized epigenetic defect exists across bladders with cancer that is not due to clonal expansion. Transurethral resection of urothelial tumors leaves behind large areas of epigenetically altered urothelium, possibly contributing to the high level of recurrence of UC. Fortunately, these hypermethylated loci may provide valuable biomarkers that have the potential to significantly impact the diagnosis and treatment of UC. Such alterations can be reversed by DNA methylation inhibitors in cell culture (49
), mouse models (15
), and white blood cells in humans (50
). Therefore it is possible that DNA methylation inhibitors may have an impact at all stages of bladder tumorigenesis: reversing premalignant epigenetic changes to prevent tumor formation or recurrence, preventing non-invasive tumors from becoming more aggressive, and inducing invasive tumors to become less malignant. In addition, the bladder may be an ideal organ for local treatment and hence avoiding systemic side-effects and highest local efficacy.