Bone marrow stroma-derived PGE2 protects BCP-ALL cells from DNA damage-induced p53 accumulation and cell death

Background B cell precursor acute lymphoblastic leukaemia (BCP-ALL) is the most common paediatric cancer. BCP-ALL blasts typically retain wild type p53, and are therefore assumed to rely on indirect measures to suppress transformation-induced p53 activity. We have recently demonstrated that the second messenger cyclic adenosine monophosphate (cAMP) through activation of protein kinase A (PKA) has the ability to inhibit DNA damage-induced p53 accumulation and thereby promote survival of the leukaemic blasts. Development of BCP-ALL in the bone marrow (BM) is supported by resident BM-derived mesenchymal stromal cells (MSCs). MSCs are known to produce prostaglandin E2 (PGE2) which upon binding to its receptors is able to elicit a cAMP response in target cells. We hypothesized that PGE2 produced by stromal cells in the BM microenvironment could stimulate cAMP production and PKA activation in BCP-ALL cells, thereby suppressing p53 accumulation and promoting survival of the malignant cells. Methods Primary BCP-ALL cells isolated from BM aspirates at diagnosis were cocultivated with BM-derived MSCs, and effects on DNA damage-induced p53 accumulation and cell death were monitored by SDS-PAGE/immunoblotting and flow cytometry-based methods, respectively. Effects of intervention of signalling along the PGE2-cAMP-PKA axis were assessed by inhibition of PGE2 production or PKA activity. Statistical significance was tested by Wilcoxon signed-rank test or paired samples t test. Results We demonstrate that BM-derived MSCs produce PGE2 and protect primary BCP-ALL cells from p53 accumulation and apoptotic cell death. The MSC-mediated protection of DNA damage-mediated cell death is reversible upon inhibition of PGE2 synthesis or PKA activity. Furthermore our results indicate differences in the sensitivity to variations in p53 levels between common cytogenetic subgroups of BCP-ALL. Conclusions Our findings support our hypothesis that BM-derived PGE2, through activation of cAMP-PKA signalling in BCP-ALL blasts, can inhibit the tumour suppressive activity of wild type p53, thereby promoting leukaemogenesis and protecting against therapy-induced leukaemic cell death. These novel findings identify the PGE2-cAMP-PKA signalling pathway as a possible target for pharmacological intervention with potential relevance for treatment of BCP-ALL

Multi-Level Interactions between the Nuclear Receptor TRα1 and the WNT Effectors β-Catenin/Tcf4 in the Intestinal Epithelium

Intestinal homeostasis results from complex cross-regulation of signaling pathways; their alteration induces intestinal tumorigenesis. Previously, we found that the thyroid hormone nuclear receptor TRα1 activates and synergizes with the WNT pathway, inducing crypt cell proliferation and promoting tumorigenesis. Here, we investigated the mechanisms and implications of the cross-regulation between these two pathways in gut tumorigenesis in vivo and in vitro. We analyzed TRα1 and WNT target gene expression in healthy mucosae and tumors from mice overexpressing TRα1 in the intestinal epithelium in a WNT-activated genetic background (vil-TRα1/Apc mice). Interestingly, increased levels of β-catenin/Tcf4 complex in tumors from vil-TRα1/Apc mice blocked TRα1 transcriptional activity. This observation was confirmed in Caco2 cells, in which TRα1 functionality on a luciferase reporter-assay was reduced by the overexpression of β-catenin/Tcf4. Moreover, TRα1 physically interacted with β-catenin/Tcf4 in the nuclei of these cells. Using molecular approaches, we demonstrated that the binding of TRα1 to its DNA target sequences within the tumors was impaired, while it was newly recruited to WNT target genes. In conclusion, our observations strongly suggest that increased β-catenin/Tcf4 levels i) correlated with reduced TRα1 transcriptional activity on its target genes and, ii) were likely responsible for the shift of TRα1 binding on WNT targets. Together, these data suggest a novel mechanism for the tumor-promoting activity of the TRα1 nuclear receptor

Functional relations between the thyroïd hormones and WNT pathways in the intestinal physiopathology : study of sFRP2 function

L'épithélium intestinal est un tissu en constant renouvellement, grâce à des cellules souches somatiques présentes dans les cryptes intestinales. Le renouvellement perpétuel et l’homéostasie de ce tissu sont assurés par plusieurs réseaux de signalisation. Il est maintenant admis que la dérégulation de ces mêmes voies est impliquée dans le processus d’initiation et/ou de progression tumorale. Mon laboratoire a décrit l'implication des hormones thyroïdiennes (HT) et de leur récepteur nucléaire TRα1 dans le contrôle de l'homéostasie intestinale, via la régulation de la voie Wnt, jouant un rôle clé dans la physiopathologie de ce tissu. Plus précisément, TRα1 active l’expression et la stabilisation de β-caténine via un mécanisme impliquant le facteur sFRP2. Au cours de ma thèse, j’ai participé, d’une part à l’étude de souris transgéniques surexprimant TRα1 dans l’épithélium intestinal et à l’analyse des mécanismes moléculaires de la régulation croisée entre TRα1 et la voie Wnt canonique dans l’induction des tumeurs intestinales. Nous avons donc démontré un rôle oncogénique de TRα1 dans l’épithélium intestinal. De plus, le mécanisme moléculaire et fonctionnel implique les deux effecteurs de la voie canonique, β-caténine et TCF4. D’autre part, j’ai analysé la fonction de sFRP2 dans la physiopathologie intestinale, et son action sur la voie Wnt. D’une manière intéressante, l’étude de la fonction de sFRP2 nous a permis de révéler son rôle original dans la différenciation des cytotypes épithéliaux. De plus, nous avons montré que sFRP2 est un modulateur positif des voies Wnt canonique et non canonique (JNK). Par ailleurs, l’absence d’expression de sFRP2 a pour conséquence d’augmenter l’apoptose dans les cryptes intestinales et ainsi diminuer le nombre de tumeurs chez des animaux double mutants sFRP2-/-/Apc+/1638N comparé aux simple mutants Apc+/1638N. Ces résultats fournissent des éléments originaux et importants sur les relations fonctionnelles entre les voies des HT et Wnt.The intestinal epithelium is a tissue constantly renewing through somatic stem cells located within the crypts. Several signalling pathways control this process and the homeostasis in this tissue. It is now recognized that the deregulation of these pathways is involved in the process of initiation and/or progression of intestinal tumors. My laboratory has described the involvement of thyroid hormones (TH) and their nuclear receptor TRα1 in the control of the intestinal homeostasis via the regulation of Wnt pathway, which plays a key role in the intestinal physiopathology. Specifically, TRα1 directly activates the expression of β-catenin and controls its stabilization through a mechanism involving sFRP2 (secreted frizzled-related protein 2). During my thesis, I participated to the characterization of transgenic mice overexpressing TRα1 in the intestinal epithelium. Moreover, I have been involved in the study of the molecular mechanisms of the cross-regulation between TRα1 and the canonical Wnt in the induction of intestinal tumors. We have therefore demonstrated an oncogenic role of TRα1 in the intestinal epithelium. In addition, the molecular and functional mechanisms involve both effectors of the canonical pathway, β-catenin and TCF4. On the other hand, I carried out the study of sFRP2 function in the intestinal physiopathology, and its action on the Wnt pathway. My data strongly suggest that sFRP2 plays an essential role in the differentiation of epithelial cytotypes. In addition, we showed that sFRP2 is a positive modulator of the canonical and non-canonical (JNK) Wnt. For instance, the absence of sFRP2 expression increases the apoptosis in the intestinal crypts and thus reduces the number of tumors in the double mutant sFRP2-/-/Apc+/1638N compared to simple mutant Apc+/1638N. These results provided original and important data of the functional relationships between TH and Wnt pathways

Relations fonctionnelles entre les voies des hormones thyroïdiennes et WNT dans la physiopathologie intestinale (étude de la fonction de sFRP2)

L'épithélium intestinal est un tissu en constant renouvellement, grâce à des cellules souches somatiques présentes dans les cryptes intestinales. Le renouvellement perpétuel et l homéostasie de ce tissu sont assurés par plusieurs réseaux de signalisation. Il est maintenant admis que la dérégulation de ces mêmes voies est impliquée dans le processus d initiation et/ou de progression tumorale. Mon laboratoire a décrit l'implication des hormones thyroïdiennes (HT) et de leur récepteur nucléaire TRa1 dans le contrôle de l'homéostasie intestinale, via la régulation de la voie Wnt, jouant un rôle clé dans la physiopathologie de ce tissu. Plus précisément, TRa1 active l expression et la stabilisation de b-caténine via un mécanisme impliquant le facteur sFRP2. Au cours de ma thèse, j ai participé, d une part à l étude de souris transgéniques surexprimant TRa1 dans l épithélium intestinal et à l analyse des mécanismes moléculaires de la régulation croisée entre TRa1 et la voie Wnt canonique dans l induction des tumeurs intestinales. Nous avons donc démontré un rôle oncogénique de TRa1 dans l épithélium intestinal. De plus, le mécanisme moléculaire et fonctionnel implique les deux effecteurs de la voie canonique, b-caténine et TCF4. D autre part, j ai analysé la fonction de sFRP2 dans la physiopathologie intestinale, et son action sur la voie Wnt. D une manière intéressante, l étude de la fonction de sFRP2 nous a permis de révéler son rôle original dans la différenciation des cytotypes épithéliaux. De plus, nous avons montré que sFRP2 est un modulateur positif des voies Wnt canonique et non canonique (JNK). Par ailleurs, l absence d expression de sFRP2 a pour conséquence d augmenter l apoptose dans les cryptes intestinales et ainsi diminuer le nombre de tumeurs chez des animaux double mutants sFRP2-/-/Apc+/1638N comparé aux simple mutants Apc+/1638N. Ces résultats fournissent des éléments originaux et importants sur les relations fonctionnelles entre les voies des HT et Wnt.The intestinal epithelium is a tissue constantly renewing through somatic stem cells located within the crypts. Several signalling pathways control this process and the homeostasis in this tissue. It is now recognized that the deregulation of these pathways is involved in the process of initiation and/or progression of intestinal tumors. My laboratory has described the involvement of thyroid hormones (TH) and their nuclear receptor TRa1 in the control of the intestinal homeostasis via the regulation of Wnt pathway, which plays a key role in the intestinal physiopathology. Specifically, TRa1 directly activates the expression of b-catenin and controls its stabilization through a mechanism involving sFRP2 (secreted frizzled-related protein 2). During my thesis, I participated to the characterization of transgenic mice overexpressing TRa1 in the intestinal epithelium. Moreover, I have been involved in the study of the molecular mechanisms of the cross-regulation between TRa1 and the canonical Wnt in the induction of intestinal tumors. We have therefore demonstrated an oncogenic role of TRa1 in the intestinal epithelium. In addition, the molecular and functional mechanisms involve both effectors of the canonical pathway, b-catenin and TCF4. On the other hand, I carried out the study of sFRP2 function in the intestinal physiopathology, and its action on the Wnt pathway. My data strongly suggest that sFRP2 plays an essential role in the differentiation of epithelial cytotypes. In addition, we showed that sFRP2 is a positive modulator of the canonical and non-canonical (JNK) Wnt. For instance, the absence of sFRP2 expression increases the apoptosis in the intestinal crypts and thus reduces the number of tumors in the double mutant sFRP2-/-/Apc+/1638N compared to simple mutant Apc+/1638N. These results provided original and important data of the functional relationships between TH and Wnt pathways.LYON-ENS Sciences (693872304) / SudocSudocFranceF

Thyroid hormone's action on progenitor/stem cell biology: New challenge for a classic hormone?

Background: Thyroid hormones are involved in developmental and homeostatic processes in several tissues. Their action results in different outcomes depending on the developmental stage, tissue and/or cellular context. Interestingly, their pleiotropic roles are conserved across vertebrates. It is largely documented that thyroid hormones act via nuclear receptors, the TRs, which are transcription factors and whose activity can be modulated by the local availability of the hormone T3. In the "classical view", the T3-induced physiological response depends on the expression of specific TR isoforms and the iodothyronine deiodinase selenoenzymes that control the local level of T3, thus TR activity. Scope of the Review: Recent data have clearly established that the functionality of TRs is coordinated and integrated with other signaling pathways, specifically at the level of stem/progenitor cell populations. Here, we summarize these data and propose a new and intriguing role for thyroid hormones in two selected examples. Major Conclusions: In the intestinal epithelium and the retina, TRα1 and TRβ2 are expressed at the level of the precursors where they induce cell proliferation and differentiation, respectively. Moreover, these different functions result from the integration of the hormone signal with other intrinsic pathways, which play a fundamental role in progenitor/stem cell physiology. General Significance: Taken together, the interaction of TRs with other signaling pathways, specifically in stem/progenitor cells, is a new concept that may have biological relevance in therapeutic approaches aimed to target stem cells such as tissue engineering and cancer. This article is part of a Special Issue entitled Thyroid hormone signalling. © 2012 Elsevier B.V. All rights reserved.SCOPUS: re.jinfo:eu-repo/semantics/publishe

The overexpression of the putative gut stem cell marker Musashi-1 induces tumorigenesis through Wnt and Notch activation

International audienceThe RNA-binding protein Musashi-1 (Msi1) has been proposed as a marker of intestinal epithelial stem cells. These cells are responsible for the continuous renewal of the intestinal epithelium. Although the function of Msi1 has been studied in several organs from different species and in mammalian cell lines, its function and molecular regulation in mouse intestinal epithelium progenitor cells are still undefined. We describe here that, in these cells, the expression of Msi1 is regulated by the canonical Wnt pathway, through a mechanism involving a functional Tcf/Lef binding site on its promoter. An in vitro study in intestinal epithelium primary cultures showed that Msi1 overexpression promotes progenitor proliferation and activates Wnt and Notch pathways. Moreover, Msi1-overexpressing cells exhibit tumorigenic properties in xenograft experiments. These data point to a positive feedback loop between Msi1 and Wnt in intestinal epithelial progenitors. They also suggest that Msi1 has oncogenic properties in these cells, probably through induction of both the Wnt and Notch pathways

Cooperation Between the Thyroid Hormone Receptor TR alpha 1 and the WNT Pathway in the Induction of Intestinal Tumorigenesis

International audienceBACKGROUND & AIMS: Colorectal tumorigenesis is a multistep process involving the alteration of oncogenes and tumor suppressor genes, leading to the deregulation of molecular pathways that govern intestinal homeostasis. We have previously shown that the thyroid hormone receptor alpha 1 (TR alpha 1) controls intestinal development and homeostasis through the WNT pathway. More precisely, TR alpha 1 directly enhances the transcription of several components of this pathway, allowing increased expression of beta-catenin/Tcf4 target genes and stimulation of cell proliferation. Because the WNT pathway is a major player in controlling intestinal homeostasis, we addressed whether the TR alpha 1 receptor has tumor-inducing potential. Methods: We generated mice overexpressing TR alpha 1 specifically in the intestinal epithelium in a wild-type (vil-TR alpha 1) or a WNT-activated (vil-TR alpha 1/Apc(+/1638N)) genetic background. RESULTS: The intestine of vil-TR alpha 1 mice presents aberrant intestinal mucosal architecture and increased cell proliferation and develops adenoma at a low rate. However, TR alpha 1 overexpression is unable to induce cancer development. On the contrary, we observed accelerated tumorigenesis in vil-TR alpha 1/Apc(+/1638N) mice compared with the Apc(+/1638N) mutants. CONCLUSION: Our results suggest that this phenotype is due to cooperation between the activated TR alpha 1 and WNT pathways. This is the first report describing the tumor-inducing function of TR alpha 1 in the intestine

The Wnt3a ligand is not sufficient to impair TRα1 transcriptional activity ex vivo.

<p>The primary cultures of intestinal epithelial cells were treated with 10 ng/ml of Wnt3a and/or 10⁻⁷ M of T3 for 24 hours. (A) The number of proliferating cells in the different experimental conditions was analyzed by Ki67 immunolabeling; all of the nuclei were labeled by Hoechst. The percentage of Ki67-positive nuclei was determined by counting under a fluorescence microscope (Zeiss Axioplan). The histograms represent the summary (mean ± sd) of the scoring of specific immunolabeling in two independent experiments each conducted in triplicate (n = 50). (B, C) Analysis of β-catenin in intestinal epithelial primary cultures by immunolabelling (B) and WB (C). Cells were in control, T3, Wnt3a and T3+Wnt3a conditions as indicated. Pictures in B show the fluorescent staining of the nuclei (blue), β-catenin (red) and the merging of each simple labeling. Bar: 15 µm. For the WB (C), we used a specific antibody allowing the detection of activated non-phosphorylated β-catenin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034162#pone.0034162-Staal1" target="_blank">[54]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034162#pone.0034162-vanNoort1" target="_blank">[55]</a>. Actin was used as loading control. The image is representative of two independent experiments. Each lane represents whole protein extracts (50 µg/lane). (D–F) RT-qPCR analysis to evaluate mRNA levels of <i>Ccnd1</i>, <i>Ctnnb1</i> and <i>Sfrp2</i>. Results are from three independent experiments, each conducted in duplicate. Values represent fold change ± sd after normalization to the control condition (Ctrl). *: P<0.05, **: P<0.01, ***: P<0.001 by two-tailed Student's t-test (n = 6).</p