Loss of Cutaneous TSLP-Dependent Immune Responses Skews the Balance of Inflammation from Tumor Protective to Tumor Promoting

Inflammation can promote or inhibit cancer progression. In this study we have addressed the role of the proinflammatory cytokine thymic stromal lymphopoietin (TSLP) during skin carcinogenesis. Using conditional loss- and gain-of-function mouse models for Notch and Wnt signaling, respectively, we demonstrate that TSLP-mediated inflammation protects against cutaneous carcinogenesis by acting directly on CD4 and CD8 T cells. Genetic ablation of TSLP receptor (TSLPR) perturbs T-cell-mediated protection and results in the accumulation of CD11b(+)Gr1(+) myeloid cells. These promote tumor growth by secreting Wnt ligands and augmenting beta-catenin signaling in the neighboring epithelium. Epithelial specific ablation of beta-catenin prevents both carcinogenesis and the accumulation of CD11b(+)Gr1(+) myeloid cells, suggesting tumor cells initiate a feed-forward loop that induces protumorigenic inflammation.

The contribution of the host stress response to the pathogenesis of Pseudomonas entomophila in the Drosophila intestine

Sveta Chakrabarti

Recently, several studies done in Drosophila have revealed that efficient and rapid recovery from bacterial infection in the gut is possible only when bacterial clearance by the immune system is coordinated with repair through renewal of the epithelium damaged by infection. In this thesis, I have analyzed how the entomopathogenic bacterium Pseudomonas entomophila affects the immune response and epithelium renewal. A microarray analysis first showed that P. entomophila is recognized by the Drosophila immune system since ingestion of this bacterium stimulated the expression of antimicrobial peptide genes by the Imd pathway. In addition, stress and damage related pathways were strongly induced by P. entomophila, which correlate with its capacity to inflict severe damage. While antibacterial genes were induced in the gut following infection with P. entomophila, the immune response was not productive due to a general inhibition of translation that affected all the newly synthesized transcripts. Furthermore, the blockage of translation also inhibited the repair program by blocking the production of JAK-STAT ligands (upd3, upd2) and growth factors, which stimulate the intestinal stem cells proliferation and differentiation. I next analyzed the pathways that link cellular damage to reduction of translation. Using a genetic approach, I showed that inhibition of translation was induced by two signaling pathways: i) the phosphorylation of elongation initiation factor 2α (eIF2α) by the stress kinase GCN2 and ii) the inhibition of the TOR pathway by the AMPK kinase. Both kinases sense metabolic deprivation, suggesting that cellular damages induced by P. entomophila induce a state of “starvation”. Inhibition of translation is usually an adaptive cellular response to adjust the metabolism to the energy status of the cells. The observation that GCN2-deficient flies survived better than wild-type flies to P. entomophila indicates that pathogenesis is linked to an over-activation of stress pathways that usually help to endure the consequence of an infection. In this in vivo model of infection, I also showed that the reduction of translation was a consequence of cellular damage to the intestine caused by host-derived reactive oxygen species (through the activity of Duox) and by the direct action of a pore-forming toxin produced by the pathogen. As a consequence of this translational arrest, flies succumbed P. entomophila infection because they were unable to repair gut damage. Finally, I showed that inhibition of translation also had a strong influence on innate immune responses observed upon P. entomophila infection. The specific activation of a systemic immune response (antimicrobial peptides produced by the fat body) observed upon P. entomophila infection could be recapitulated by feeding flies with a non-lethal pathogen and an inhibitor of translation. Hence, I showed translation inhibition could be an important feature that shapes the immune response. The p38 MAPK family is involved in stress and immunity in both mammals and Drosophila. In Drosophila, three p38-MAPK-encoding genes, p38a, p38b and p38c, have been identified but their function in the gut immune response is poorly characterized. In the second part of my thesis, I have analyzed the role of these three p38 MAPKs in Drosophila intestinal immunity, especially p38c, which is strongly enriched in the gut. I first confirmed that the three p38 are involved kinases are involved in the defense to oral infection with pathogenic (but non-lethal) bacteria such as Erwinia carotovora 15. Surprisingly, p38c mutant flies were more resistant to P. entomophila and did not show the reduction in translation observed in wild-type flies. Interestingly, the transcription of the ROS producing enzyme Duox was reduced in p38c raising the hypothesis that p38 contributes to P. entomophila pathogenicity by activating Duox. Furthermore, I have obtained preliminary data indicating that p38c and the transcription factor ATF-3 act in the same pathway contributing the host immune response and lipid metabolism. Together, my thesis reveals the complex cross-talks between stress and immune pathways during microbial infection. While, it shows that stress pathways usually contribute to endure the damage caused by infection, excessive activation of these pathways can contribute to pathogenesis.

EPFL2013

90 YEARS OF PROGESTERONE Progesterone receptor signaling in the normal breast and its implications for cancer

Cathrin Brisken, Valentina Scabia

Progesterone is considered as the pregnancy hormone and acts on many different target tissues. Progesterone receptor (PR) signaling is important for normal development and the physiologic function of the breast and impinges on breast carcinogenesis. Both systemically and locally, in the breast epithelium, there are multiple layers of complexity to progesterone action, many of which have been revealed through experiments in mice. The hormone acts via its receptor expressed in a subset of cells, the sensor cells, in the breast epithelium with different signaling outcomes in individual cells eliciting distinct cell-intrinsic and paracrine signaling involving different mediators for different intercellular interactions. PR expression itself is developmentally regulated and the biological outcome of PR signaling depends on the developmental stage of the mammary gland and the endocrine context. During both puberty and adulthood PR activates stem and progenitor cells through Wnt4-driven activation of the myoepithelium with downstream Adamts18-induced changes in extracellualr matrix (ECM) / basal membrane (BM). During estrous cycling and pregnancy, the hormone drives a major cell expansion through Rankl. At all stages, PR signaling is closely tied to estrogen receptor a (ER) signaling. As the PR itself is a target gene of ER, the complex interactions are experimentally difficult to dissect and still poorly understood. Ex vivo models of the human breast and studies on biopsy samples show that major signaling axes are conserved across species. New intraductal xenograft models hold promise to provide a better understanding of PR signaling in the normal breast epithelium and in breast cancer development in the near future.

BIOSCIENTIFICA LTD2020

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.