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

The Bcl9-beta-catenin interface as a potential therapeutic target for colorectal cancer

Andreas Eduard Moor

Aberrantly active Wnt signaling is the hallmark driver of colorectal cancer (CRC). Bcl9/9l proteins are components of the main transcriptional activation complex of canonical Wnt signaling and interact with beta-catenin via a highly conserved homology domain (HD2). Intestinal loss of Bcl9 affects wound healing and Lgr5+-cancer stem cell (CSC) maintenance, but does not perturb normal tissue homeostasis. The aim of the present thesis was to validate the Bcl9-Î²-catenin interaction as a potential target for modulating Wnt signaling and abolishing intestinal stemness traits in colorectal cancer. We investigated the role of the Bcl9-beta-catenin interaction in the mouse AOM-DSS chemical carcinogenesis CRC model. Subsequently, we validated our observations in an independent transgenic APC-Kras CRC model (truncated APC tumor suppressor and mutated Kras expressed in the intestine). The expression of Bcl9 proteins was abolished exclusively in the gastrointestinal epithelium using constitutive or inducible Cre-recombinase expressed under the control of the Villin promoter. To corroborate that the loss of Bcl9 phenotype was due to the abrogation of the Bcl9-beta-catenin interaction and not to a Î²-catenin-independent function of Bcl9, we made use of an additional Bcl9 mutant mouse strain in which only the HD2 of both Bcl9 proteins was deleted, selectively preventing the Bcl9-beta-catenin interaction. The inducible deletion of Bcl9 in established AOM-DSS tumors led to a loss in stem cell, Wnt and EMT gene expression signatures, recapitulating the phenotype that we previously described when Bcl9 was deleted constitutively. A 14d partial decrease of Bcl9 proved sufficient to suppress the expression of mesenchymal marker Vimentin in a cell autonomous fashion. Importantly, AOM-DSS tumors with Bcl9 proteins selectively lacking the HD2 beta-catenin interaction domain displayed the same phenotype as observed in tumors that entirely lacked Bcl9 proteins. Bcl9-knockout tumors induced in the Wnt active APC-Kras tumor model were compared with their wild-type counterparts. Wild-type APC-Kras mutant tumors displayed fewer mesenchymal traits when compared to AOM-DSS induced tumors. Global gene expression analysis by RNA sequencing confirmed a strong positive correlation between Bcl9/9l-dependent transcriptional changes in both independent tumor models. Genes differentially expressed between wild-type and Bcl9/9l ablated tumors were used to probe CRC databases for correlation with patient outcome and revealed a strong association with vastly improved disease-free and overall survival, independent of clinical and molecular parameters. We have shown that preventing the interaction of Bcl9 with beta-catenin in established tumors in Wnt-activated CRC models rapidly resulted in the loss of stemness and mesenchymal traits and concomitant induction of a more differentiated phenotype. Importantly, these loss-of-Bcl9 traits are prognostic for favorable outcome in CRC patients and we have not observed any overt anomalies upon intestinal ablation of Bcl9 in adult mice. Overall, our observations indicate that targeting the Bcl9-beta-catenin interaction in Wnt-activated tumors might be well tolerated and a generally applicable pharmacological strategy to target CSCs to potentially prevent tumor recurrence and improve progression free survival.

EPFL2014

Role of Bcl9 and Bcl9l in Homeostasis, Regeneration and Tumorigenesis of the Gastrointestinal Epithelium

Norbert Wiedemann

Wnt signaling is essential for embryogenesis and adult tissue homeostasis and contributes to cancer development, especially in the colon. A new essential pathway component, legless, was identified in Drosophila melanogaster. Mice with Cre/loxP-mediated conditional ablation of the legless homologues Bcl9 and Bcl9l in the intestinal epithelium were generated to assess the contribution of the genes to Wnt signaling and their potential suitability as therapeutic drug targets. Initial analysis had revealed that both genes were not required for self-renewal of the intestinal epithelium during homeostasis, but for regeneration of experimentally induced colon wounds. Furthermore, formation of chemically induced colon tumors was not impaired and Bcl9/Bcl9-mutant tumors were morphologically indistinguishable from wild-type tumors and displayed hallmarks of active Wnt signaling. This study aimed at further investigating the molecular processes involving Bcl9 and Bcl9l in wound regeneration and tumorigenesis in the colon, and in particular relatedness of the genes to Wnt signaling. To this end, transcriptomes of laser microdissected wound regions were analyzed by microarray-based gene expression profiling and realtime quantitative PCR, but did not reveal significant changes in gene expression at the time point chosen for analysis. However, expression profiling of homeostatic colon epithelium revealed a reduced expression of several intestinal stem cell marker genes in Bcl9/Bcl9l-mutant colon epithelium, suggesting that the regeneration deficit has been caused by functional impairment of intestinal stem cells. This stem cell deficiency did not differ between young and old mice and was also weakly detectable in small intestinal epithelium. To verify that tumors originated from cells in which complete deletion of all four Bcl9 and Bcl9l alleles had occurred and to assess Wnt signaling activity more comprehensively, laser microdissected tumor regions were profiled using microarrays and realtime quantitative PCR. This analysis confirmed that Bcl9 and Bcl9l were not required for colon tumorigenesis and revealed that despite the morphological similarity, transcriptional profiles of Bcl9/Bcl9l-mutant tumors were very different from wild-type tumors. Notably, loss of Bcl9 and Bcl9l caused reduced expression of a subset of direct Wnt target genes, genes related to epithelial-mesenchymal transition (EMT) and intestinal stem cell traits. While wild-type tumor epithelium appeared to be in a metastable state, characterized by co-expression of epithelial and mesenchymal traits, Bcl9/Bcl9l-mutant tumor epithelium predominantly expressed epithelial traits. In addition, a marked reduction in expression of several genes associated with malignant tumor properties was observed in Bcl9/Bcl9l-mutant tumors. This was highlighted by the preservation of an intact basement membrane around Bcl9/Bcl9l-mutant tumor epithelium, which was absent in wild-type tumors, and indicated that loss of Bcl9/Bcl9l decreased tumor aggressiveness. Of note, EMT and stemness traits are correlated to tumor resistance to therapy and thus interfering with Bcl9/Bcl9l function might restore drug responsiveness. As a proof of concept, it was shown that the malignant traits mediated by Bcl9/Bcl9l could be abrogated in wild-type tumors by inducing deletion of the genes. These findings suggest that targeting Bcl9 and Bcl9l with therapeutic drugs has the potential to attenuate the malignancy of colon tumors.

EPFL2011

The use of RNA interference to increase PEI-mediated transient gene expression in mammalian cells

Martin Bertschinger

DNA uptake by polyethylenimine (PEI)-mediated transfection was investigated in Chinese hamster ovary (CHO DG44) cells. Rapid DNA uptake was observed with the maximum occurring during the first 45-60 min after addition of PEI-DNA complexes to cells. With longer incubation times, the aggregation of PEI-DNA particles reduced the rate of DNA uptake. At early times after transfection, the kinetics of DNA uptake were constant, suggesting that the limiting factor to PEI transfection was the rate of endocytosis. The most efficient transfection with PEI was observed at a density of 2 x 106 cells/ml and at a PEI:DNA ratio of 2:1 (w/w). DNA uptake at higher PEI:DNA ratios was also efficient, but the toxicity of PEI decreased the recombinant protein yield. The optimal PEI:DNA ratio was found to be related to the number of cells in the culture. Once in the cell, PEI:DNA particles must be disassembled to allow transcription of the recombinant gene. Here their disassembly was investigated using a simple in vitro assay. The particles were formed with either a linear (25 kDa or JetPEITM) or a branched PEI (2, 25, or 750 kDa) and then mixed with varying amounts of a competitor (RNA, DNA, or heparin). The amount of plasmid DNA released from particles was determined by agarose gel electrophoresis or spectroscopy. The stability of the particles to changes in pH, osmolarity, and the PEI:DNA ratio was also determined. Either the presence of heparin or high salt concentrations (1-2 M) yielded complete particle disassembly for all PEIs tested. The addition of RNA to particles formed with linear PEIs or branched 2 kDa PEI resulted in the rapid release of all the plasmid DNA, even at an RNA concentration of 50 µg/ml. However, the presence of RNA at concentrations up to 400 µg/ml induced only partial disassembly of particles formed with branched PEIs of 25 or 750 kDa. In the presence of competitor DNA, slow particle disassembly was observed with particles made with linear 25 kDa PEI, JetPEI, and branched 2 kDa PEI, but DNA release was not seen for particles made with branched PEIs of higher molecular weight. The presence of BSA only resulted in partial disassembly of PEI-DNA particles, and DNA release was not observed after the exposure of particles to acidic pH as low as 3. For all the PEIs tested, their stability increased as the PEI:DNA ratio increased. It was concluded from these studies that branched PEIs have a higher affinity for DNA than linear PEIs and that the intracellular disassembly of PEI-DNA particles may involve interactions between PEI and cellular RNA. In the second part of the work, RNA interference (RNAi) was used to enhance transient gene expression by knockdown of two different mRNAs, the E1A mRNA in human embryonic kidney 293 EBNA (HEK293E) cells and the ube2i mRNA in CHO DG44 cells and HEK293 cells. HEK293 cells are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNAi. The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1α (EF-1α) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to 3-fold. E1A mRNA depletion also resulted in a 2-fold increase in transient expression of a recombinant IgG in suspension cultures when the IgG light and heavy chain genes were driven by the EF-1α promoter. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells. Efficient RNAi-mediated depletion of ube2i mRNA in CHO DG44 cells was accomplished by co-expression of 2 shRNAs targeting two distinct sides in the ube2i mRNA, whereas knockdown with only one of the shRNAs at a time was insufficient for reducing ube2i mRNA levels. The reduction of the ube2i mRNA level in CHO DG44 cells was shown to enhance transient GFP expression 2-fold and IgG expression 2.5-fold. For these experiments, the GFP gene was under the control of the CMV IE promoter and the IgG light and heavy chain genes were under the control of the human EF-1α promoter. Thus, the enhancement of transient gene expression resulting from ube2i mRNA depletion was not promoter-dependent. These results demonstrated that sumoylation has a negative effect on transient gene expression in CHO DG44 and HEK293E cells.

EPFL2006