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

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

HCMV pUL135 remodels the actin cytoskeleton to impair immune recognition of infected cells

Immune evasion genes help human cytomegalovirus (HCMV) establish lifelong persistence. Without immune pressure, laboratory-adapted HCMV strains have undergone genetic alterations. Among these, the deletion of the UL/b’ domain is associated with loss of virulence. In a screen of UL/b’, we identified pUL135 as a protein responsible for the characteristic cytopathic effect of clinical HCMV strains that also protected from natural killer (NK) and T cell attack. pUL135 interacted directly with abl interactor 1 (ABI1) and ABI2 to recruit the WAVE2 regulatory complex to the plasma membrane, remodel the actin cytoskeleton and dramatically reduce the efficiency of immune synapse (IS) formation. An intimate association between F-actin filaments in target cells and the IS was dispelled by pUL135 expression. Thus, F-actin in target cells plays a critical role in synaptogenesis, and this can be exploited by pathogens to protect against cytotoxic immune effector cells. An independent interaction between pUL135 and talin disrupted cell contacts with the extracellular matrix

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

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Solute carriers (SLCs) in cancer

During tumor progression cells acquire an altered metabolism, either as a cause or as a consequence of an increased need of energy and nutrients. All four major classes of macromolecules are affected: carbohydrates, proteins, lipids and nucleic acids. As a result of the changed needs, solute carriers (SLCs) which are the major transporters of these molecules are differently expressed. This renders them important targets in the treatment of cancer. Blocking or activating SLCs is one possible therapeutic strategy. For example, some SLCs are upregulated in tumor cells due to the increased demand for energy and nutritional needs. Thus, blocking them and turning off the delivery of fuel or nutrients could be one way to interfere with tumor progression. Specific drug delivery to cancer cells via transporters is another approach. Some SLCs are also interesting as chemosensitizing targets because blocking or activating them may result in an altered response to chemotherapy. In this review we summarize the roles of SLCs in cancer therapy and specifically their potential as direct or indirect targets, as drug carriers or as chemosensitizing targets. (C) 2012 Elsevier Ltd. All rights reserved

ß-catenin binds to the promoter region of FOXQ1 and increase transcription.

<p>(<b>A</b>) ChIP assay was performed on SW480 cells with antibodies against ß-catenin and IgG (control). Input and immunoprecipitated DNA was measured by qRT-PCR using primers amplifying the promoter region of FOXQ1 and the −100/0 promoter region of GAPDH. Data are mean ± SD n = 3, **P<0.01 using Mann-Whitney test. (<b>B</b>) Graphical depiction of the promoter region of FOXQ1, and the generated promoter reporter vectors. (<b>C</b>) Luciferase assay showing the effect of Wnt activation in HEK293 and HCT116 cells (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060051#pone-0060051-g005" target="_blank">figure 5</a>) transfected with the wild type promoter region of FOXQ1 (pGL3_FOXQ1_WT), a mutated TCF4 binding site (pLG3_FOXQ1_Mut) or an empty vector (pGL3_Emtpy). Data are mean ± SD n = 4–7, *P<0.05, ***P<0.001 by the Kruskal-Wallis Conover test.</p

FOXQ1 is induced on protein level in human colon carcinoma as compared to normal tissue.

<p>Western blot analysis of FOXQ1 in human CRC biopsy samples from three different patients. GAPDH was used as loading control. T = tumor tissue, N = normal colon tissue.</p

FOXQ1 expression correlates with the average Wnt signature strength in a wide panel of cell lines.

<p>Heatmap showing the correlation coefficients between single genes and the mean expression of all 24 direct Wnt targets in various cancer cell lines (data set GSE36133). Colon n = 57, breast n = 58, lung = 174, pancreas n = 44, stomach n = 38, all n = 967.</p

Activation of the Wnt pathway leads to increased FOXQ1 expression.

<p>(<b>A</b>) qRT-PCR analysis of FOXQ1 expression in HEK293 and HCT116 cell lines after Wnt activation. Cell were treated with 20 mM LiCl for 24 hours or transfected with a constitutively active form of ß-catenin (S33Y). Data are mean ± SD n = 3, **P<0.01 using Mann-Whitney test. (<b>B</b>) Western blot of FOXQ1 in HEK293 and HCT116 after Wnt activation as described above. GAPDH was used a loading control. (<b>C</b>) HEK293 and HCT116 cells stably expressing the Wnt sensitive reporter 7TGC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060051#pone.0060051-Fuerer1" target="_blank">[25]</a> were treated as described above and imaged. GFP (green) is under the control of a Wnt sensitive promoter and mCherry (red) is constitutively expressed to identify infected cells, white bar indicates 20 µm.</p

Correlation of FOXQ1 with the Wnt pathway.

<p>Gene set enrichment analysis of FOXQ1 with a selection of gene set related to Wnt signalling. GSEA preformed in various solid tumors showed a significant enrichment when using direct Wnt targets and gene sets related to Wnt activation. SIZE = size of tested gene set, ES = enrichment score, NOM p-val = Nominal p-value, FDR q-val = false discovery rate q-value.</p