Title: Molecular charateristics of desmoid tumors
Other Titles: Moleculaire karakteristieken van desmoid tumoren
Authors: Amini Nik, Saeid; M0131907
Issue Date: 15-Oct-2007
Abstract: Desmoid tumors (aggressive fibromatosis) are locally invasive soft tissue tumors in which b-Catenin/TCF3 mediated Wnt signaling is activated. More than 80% of the desmoid tumors contain activating mutations in b-Catenin. Here, we tried to identify other possible pathways or molecules which might be involved in their pathogenesis.In order to evaluate the Wnt component fingerprint in desmoids, we subjected desmoid RNA to Wnt components arrays. We showed that despite the oncogenic events in desmoid tumors which increase the level of ß-Catenin at the protein level, the canonical and non-canonical Wnt pathways were activated at the ligand level and they were functional. The results showthat Wnt5a, Wnt6 and Wnt8a are upreguated up to 10 fold in desmoids compare to the fascia of the same patient. ß-Catenin at the RNA level was increased up to 10 fold. Also, active ß-Catenin protein and alpha smooth muscle actin were upregulated up to 3.3 and 28 fold respectively. Dkk1 blocked the canonical Wnt pathway. It increased the level of alpha smoothmuscle actin 4.5 fold, decreased BrdU incorporation of desmoid cells down to 50%, delayed healing of scratched desmoid cultures and decreased the level of nuclear active and total ß-Catenin. The expression and function of Wnt5a was further examined. The results show that blocking the canonical Wnt pathway does not change the level of Wnt5a. Wnt5a had a pro-proliferative effect in desmoid cells. In chapter 2, we showed that despite the oncogenic mutations in ß-Catenin, TGF-ß was a modulator of the ß-Catenin levels in tumoral fibroblastsas well as in non tumoral fibroblasts. The results showed that the TGF-ß pathway was active in desmoids cells and in in situ tumors. A dose dependent increase in ß-Catenin protein levels was observed after TGF-ß treatment in combination with an increased repression of GSK-3β both in normal and tumoral fibroblasts. TGF-ß stimulation also led to an altered – up to 5 fold- transcriptional activity of ß-Catenin responsive promoters, such as IGFBP6 as well as an increase of TOPflash activity. TGF-ßstimulation increased cell proliferation and BrdU incorporation 2.5 times. Taken together, we proposed in this chapter that TGF-ß is a modulator of the ß-Catenin levels in tumoral and non tumoral fibroblasts, despite the oncogenic mutations already present in this gene in the myofibroblasts of desmoid tumors. This modulation of ß-Catenin levels by TGF-ß may be involved in determining the tumoral phenotype of the cells.Because, the Wnt signaling pathway interacts with the tumor suppressor gene WT1 in normal kidney development and plays a role in the genesisof some Wilms tumors, we were wondered if this relation applied to desmoids.To investigate whether this hypothesis could be extended to desmoids, in chapter 3 we searched for WT1 mutations and studied WT1 expression in b-Catenin mutant desmoid tumors. The results showed medium to high abundant levels of WT1 mRNA expression in all tested desmoids cells, while adjacent normal fibroblasts showed low expression of WT1 . Western blot analysis and immunohistochemistry confirmed this overexpression at the protein level. A mutational screen of the WT1 zinc-finger region by sequence analysis did not identify any mutations. Finally, we investigated a possible role of b-Catenin on WT1 regulation and vice versa. Overexpression of different b-Catenin mutants in the HEK293T cell line did not modulate WT1 promoter activity and WT1 did not affect b-Catenin/TCF transcriptional activity in this cell line. Our results show that the wild-type WT1 gene is strongly overexpressed in b-Catenin mutant desmoid tumors and may play a role in tumorigenesis of desmoid tumors, similar to what has been suggested in some epithelial malignancies.Desmoid tumors have long puzzled clinicians because of their stop and go behavior, where regions of the tumor may grow rapidly and later stop growing. These tumors are known to contain oncogenic activating ß-Cateninmutations, a protein implicated in uncontrolled cell proliferation and migration in malignancies. In chapter 4, we asked why desmoids, harboring active Wnt and TGF-ß signaling pathways and upregulated WT1 (a proteinknown to be a tumor suppressor), have OFF and ON behaviors. Could some of these players have a dual effect on the cells which can turn the tumor on and off ? Indeed, some reports have shown a pro-apoptotic effect ofß-Catenin besides its oncogenic effect. In this chapter we studied thiseffect in mesenchymal cells which are the closest relatives of desmoid cells. Here, we showed that Wnt pathway stimulation can induce both pro-proliferative and pro-apoptotic effects in various mesenchymal cells. This effect correlated with the levels of active ß-Catenin in these cells. Treatment of normal human fibroblasts as well as mouse fibroblasts and C2C12 cells with high doses of either Wnt3a conditioned medium or LiCl decreased the number of viable cells by 75% and increased caspase 3/7 activity up to 32% suggesting a pro-apoptotic role of high levels of ß-Catenin. This effect was not seen in transformed NIH3T3 cells or CHO cells. Using an apoptosis array, measuring the expression of 84 genes related to apoptosis at the RNA level, revealed that high levels of Wnt pathway activity induced both the intrinsic and extrinsic apoptosis pathways. In contrast low doses of Wnt pathway inducers increased the number ofviable cells up to 50% confirming the pro-proliferative effect of ß-Catenin. Taken together in this manuscript we showed some new molecular characteristic of desmoid tumors, which increases the potential number of candidates involved in the pathogenesis of desmoids but on the other hand might be targets for new therapeutic approaches.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Human Mutations and Polymorphisms Section (-)
Translational Research in GastroIntestinal Disorders

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