FOXQ1, a Novel Target of the Wnt Pathway and a New Marker for Activation of Wnt Signaling in Solid Tumors

Pascale Anderle Pedone, Thierry Sengstag
Public Library of Science, 2013
Article

Résumé

Background: The forkhead box transcription factor FOXQ1 has been shown to be upregulated in colorectal cancer (CRC) and metastatic breast cancer and involved in tumor development, epithelial-mesenchymal transition and chemoresistance. Yet, its transcriptional regulation is still unknown. Methods: FOXQ1 mRNA and protein expression were analysed in a panel of CRC cell lines, and laser micro-dissected human biopsy samples by qRT-PCR, microarray GeneChip (R) U133 Plus 2.0 and western blots. FOXQ1 regulation was assayed by chromatin immunoprecipitation and luciferase reporter assays. Results: FOXQ1 was robustly induced in CRC compared to other tumors, but had no predictive value with regards to grade, metastasis and survival in CRC. Prototype-based gene coexpression and gene set enrichment analysis showed a significant association between FOXQ1 and the Wnt pathway in tumors and cancer cell lines from different tissues. In vitro experiments confirmed, on a molecular level, FOXQ1 as a direct Wnt target. Analysis of known Wnt targets identified FOXQ1 as the most suitable marker for canonical Wnt activation across a wide panel of cell lines derived from different tissues. Conclusions: Our data show that FOXQ1 is one of the most over-expressed genes in CRC and a direct target of the canonical Wnt pathway. It is a potential new marker for detection of early CRC and Wnt activation in tumors of different origins.

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https://infoscience.epfl.ch/record/188903?ln=fr

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Pascale Anderle Pedone, Thierry Sengstag
Public Library of Science, 2013
Article

Résumé

Official source

https://infoscience.epfl.ch/record/188903?ln=fr

À propos de ce résultat

Concepts associés (14)

Concepts associés

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Publications associées (34)

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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

Chargement

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

Fanny Bringolf

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

2010

Chargement

Notch signaling is a conserved cell-to-cell communication pathway essential in the development and maintenance of various tissues. Upon ligand binding, Notch receptors on the cell surface are proteolytically cleaved, generating the intracellular domain of Notch (NICD) which acts as a transcriptional regulator. Signaling through Notch1 is essential for normal T cell development, and de-regulated Notch1 signaling leads to the development of acute lymphoblastic T cell leukemia (T-ALL). Mouse models of retroviral overexpression of N1ICD in hematopoietic progenitors in bone-marrow (BM) chimeric mice clearly show that Notch1 in T-ALL acts as an oncogene. MicroRNAs (miRNAs) are small RNA molecules that function as post-transcriptional negative regulators of gene expression. They have been described to play either oncogenic or tumor suppressive roles in Notch1-driven T-ALL. However, it is currently unknown whether miRNAs are essential in Notch1-driven leukemogenesis or in T-ALL maintenance. Dicer1 is an essential enzyme for the generation of mature, functional miRNAs. In this study, the global requirement for miRNAs in T-ALL was assessed via conditional ablation of Dicer1 during early N1ICD-mediated leukemogenesis and in later stages of established T-ALL in vivo. Interestingly, both T-ALL development and maintenance in N1ICD overexpressing BM chimeras were dependent on Dicer1. This was found to be due to induction of apoptosis in Dicer1-deficient T-ALL cells. Microarray-based expression analysis demonstrated that potentially oncogenic miRNAs were up-regulated in both mouse and human T-ALL samples compared with normal T cell progenitors. Among the abundantly expressed miRNAs, candidates previously linked to T-ALL were detected as well as novel candidates. Among them, miR-21, which has previously been linked to tumor biology in a variety of solid tumors, was found to be the most differentially regulated in T-ALL and early and late T cell progenitors. It was also found to be abundantly expressed in primary mouse T-ALL specimens as well as human T-ALL cell lines and primary patient T-ALL samples. Using a novel lentivirus-based miRNA activity reporter system, I demonstrated that miR-21 is active in T-ALL cell lines both in vitro and in vivo. In addition, KD of miR-21 led to apoptosis in T-ALL cells with high miR-21 activity, consistent with miR-21 target gene de-repression. Taken together, these findings demonstrate for the first time that miR-21, in the context of Notch1-driven T-ALL, acts in an oncogenic fashion. It does so by facilitating T-ALL cell survival, which is, at least in part, due to repression of programmed cell-death protein 4 (Pdcd4), a miR-21 target gene and potential novel tumor suppressor in T-ALL. In summary, this study shows that in Notch1-driven T-ALL, miRNAs play an essential role by facilitating T-ALL cell survival, and that miR-21 is a novel oncogenic miRNA in this disease.

EPFL2014

Chargement

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

Andreas Eduard Moor

EPFL2014

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

Fanny Bringolf

2010

EPFL2014