Aberrant splicing of the E-cadherin gene. Chronic lymphocytic leukemia (CLL) is the most common leukemia and an incurable disease. To identify novel genetic mutations in chronic lymphocytic leukemia cells, we used a microarray technique to screen for genes, which harbor premature termination codons (PTCs). These PTC mutations contribute to oncogenesis by expressing a truncated non-functional mRNA, which is degraded by the nonsense-mediated degradation (NMD) pathway resulting in loss of expression. Among the genes with highest upregulation, we identified the E-cadherin gene, a tumor suppressor gene. The PTC is in an alternatively spliced of E-cadherin transcript which is stabilized when the NMD pathway is blocked. This transcript of E-cadherin completely lacks exon 11 (exon skipping), resulting in a frameshift and a PTC codon in exon 12. RT-PCR analysis demonstrated that the exon 11 skipping occurs in normal B cells as well but at a much lower frequency. We also found a significant decrease in total wild type E-cadherin RNA expression in 62% of CLL specimens (n=35). Furthermore, there was an inverse correlation (p=0.018) between high aberrant transcript and lower E-cadherin expression in CLL specimens. Our results suggest a novel mechanism of E-cadherin gene loss of function in which the trans- factors/splicing factors in CLL cells induce an increased non-productive/aberrant splicing of this tumor suppressor gene. A downstream effect of loss of E-cadherin is the activation of the wnt pathway as beta-catenin is now able to translocate to the nucleus. This pathway is active in CLL cells and our data using reporter assays confirms this finding. Our preliminary experiments also show the reporter activity is higher in CLL specimens with lower E-cadherin expression and the reporter activity can be reversed with ectopic expression of E-cadherin alone.
In specific aim 1, we will study the significance of E-cadherin loss of expression in CLL cells. E-cadherin loss can result in activation wnt- -catenin pathway along which can also be activated by other wnt pathway activators (e.g. wnt and frizzled genes). We will determine the relative role of E-cadherin loss and the wnt/frizzled genes in wnt pathway upregulation. Experiments will be performed to inhibit this pathway by E-cadherin re-expression and its subsequent effects on apoptosis, migration and invasion.
In specific aim 2, the cis- and trans- elements involved in the aberrant splicing of exon 11 will be studied. With the help of a minigene construct, the cis-elements involved in aberrant splicing will be analyzed by deletions and an antisense oligo strategy. The trans-factors or the splicing factors involved in aberrant splicing will be identified by studying the differentially expressed splicing factors in CLL and normal B cells. The role of these splicing factors will be confirmed in minigene experiments by expressing or inactivating the factors and determining their effect on aberrant splicing. Our goal here is to identify the relevant cis-elements and the trans-factors which bind them so that attempts can be made to modify the aberrant splicing process and restore E-cadherin expression.
Aberrant splicing of the E-cadherin gene. We are studying the most common leukemia in the western world, chronic lymphocytic leukemia (CLL). The incidence of this leukemia increases with age and its incidence is higher in males. For these reasons, CLL is a common diagnosis in the Veteran patient population. Our ability to predict outcome in CLL patients is still limited and therefore, we are unable to tailor specific treatment to CLL patients. Identification of new genetic abnormalities in CLL will be helpful to determine prognosis and open new avenues for treatment. We have identified genetic abnormalities in a gene called E-cadherin in CLL which is a well known gene in other types of cancers. We have performed extensive experimental work, which supports our contention that E-cadherin is an important gene in CLL. Our proposal will further study the role of this gene in CLL.