Premature termination codons (PTCs) in tumor suppressor genes can contribute to oncogenesis. PTCs prematurely terminate transcription, leading to expression of a truncated, mRNA. The nonsense-mediated degradation (NMD) pathway degrades the aberrant transcripts resulting in loss of expression. To identify RNA transcripts with PTCs in chronic lymphocytic leukemia specimens (CLL), we employed a microarray technique to screen for genes which upregulate their RNA signal upon NMD blockade. In the list of genes with highest upregulation, we identified the E-cadherin gene, a known tumor suppressor gene. On further investigation, we found that the PTC was due to stabilization of an alternatively spliced E-cadherin transcript. This transcript completely lacks exon 11 (exon skipping), resulting in a frameshift and a PTC which is recognized by the NMD pathway and degraded. RT-PCR analysis demonstrated that the exon 11 skipping occurs in normal B cells as well but at a much lower frequency. A real time PCR based strategy to quantify the relative amount of spliced transcript confirmed a 10–30 fold increase in exon 11 skipping in CLL cells (n=29) as compared to normal B cells (n=4) and the degree of skipping increased with increased Rai stage. In addition, real time PCR also demonstrated a significant decrease in total wild type E-cadherin RNA expression in 58% of CLL specimens compared to normal B cells. As prior work demonstrates an upregulated wnt/β-catenin pathway in CLL and, and that E-cadherin can be a physiologic regulator of this pathway, we tested wnt pathway reporter expression in CLL specimens. High expression was identified and ectopic expression of E-cadherin in some samples with E-cadherin loss was sufficient to inhibit the wnt-reporter activity. This suggests that E-cadherin loss, possibly due to exon 11 alternative splicing and exon skipping plays a role in upregulated wnt signaling. As there are no mutations in exon 11 or its flanking intronic regions in CLL cells, our results suggest a novel mechanism of E-cadherin gene inactivation in which the trans-factors/splicing factors in malignant B lymphocytes induce an increased nonproductive splicing of a tumor suppressor gene.

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