Defining new criteria for selection of cell-based intestinal models using publicly available databases

Background: The criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies. Results: We made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT) and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics. Conclusions: This study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models, introducing a rank order of selected features may allow selecting model cell lines that are more adapted and pertinent to the addressed biological question.

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

A colorectal cancer classification system that associates cellular phenotype and responses to therapy

Douglas Hanahan, Krisztian Homicsko, Anguraj Sadanandam, Stephan Wullschleger

Colorectal cancer (CRC) is a major cause of cancer mortality. Whereas some patients respond well to therapy, others do not, and thus more precise, individualized treatment strategies are needed. To that end, we analyzed gene expression profiles from 1,290 CRC tumors using consensus-based unsupervised clustering. The resultant clusters were then associated with therapeutic response data to the epidermal growth factor receptor–targeted drug cetuximab in 80 patients. The results of these studies define six clinically relevant CRC subtypes. Each subtype shares similarities to distinct cell types within the normal colon crypt and shows differing degrees of 'stemness' and Wnt signaling. Subtype-specific gene signatures are proposed to identify these subtypes. Three subtypes have markedly better disease-free survival (DFS) after surgical resection, suggesting these patients might be spared from the adverse effects of chemotherapy when they have localized disease. One of these three subtypes, identified by filamin A expression, does not respond to cetuximab but may respond to cMET receptor tyrosine kinase inhibitors in the metastatic setting. Two other subtypes, with poor and intermediate DFS, associate with improved response to the chemotherapy regimen FOLFIRI1 in adjuvant or metastatic settings. Development of clinically deployable assays for these subtypes and of subtype-specific therapies may contribute to more effective management of this challenging disease.

Nature Publishing Group2013

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.