Functional analysis and knock‐down phenotype of BCL9 and BCL9L in cancer cell lines

Therapy‐resistant tumor cells often display EMT and stem cell‐like traits. The canonical Wnt pathway has been associated with these phenotypes in many cancer types. Our group has shown in mice that two proteins of this pathway, Bcl9 and Bcl9l, participate in controlling Wnt-mediated stem cell traits. We hypothesized that inhibition of BCL9/BCL9L could be a potential strategy to revert cells with stem‐cell properties to a differentiated epithelial phenotype, and thus also re‐establish their sensitivity to chemotherapy. Preliminary experiments in a human colon cancer cell line showed that BCL9L single knock‐down (KD) surprisingly already completely abrogated expression of selected Wnt target genes, despite the presence of the second paralog BCL9, which was even up‐regulated upon loss of BCL9L. This observation raised the question whether the remaining BCL9 protein might be dysfunctional, or whether BCL9 and BCL9L are, against our expectation, not functionally redundant. Thus, we characterized Bcl9 expression at transcriptional and genomic level by reverse transcription and DNA sequencing. By sequencing, we identified three different BCL9 mRNA species in SW480 cells (wild‐type (WT) and two isoforms carrying short deletions), which we also confirmed to be present in other carcinoma cell lines. No abnormalities were found on the genomic level. We then assessed Bcl9 and Bcl9l expression at protein level using standard Western blotting. In the different cell lines the proteins showed distinct molecular weights, which may correspond to different posttranslational modifications that could be associated with different functions. To generally explore BCL9 function, we silenced the gene using lentivirus vector‐based expression of shRNAs. Further analysis of the observed Bcl9 KD on a broad panel of Wnt target genes and comparison with the Bcl9l KD phenotype will help elucidating Bcl9 and Bcl9L function