Lysyl Oxidase silencing impairs keratinocyte differentiation in a reconstructed-epidermis model

International audienceLysyl Oxidase (LOX) is an extracellular enzyme involved in the maturation of connective tissues. It also acts in many cell types as a regulator of cell behaviour and phenotype through intracellular signalling pathways. Recently, LOX was shown to be present in human epidermis where its precise functions remain unclear. We showed here that in confluent monolayer cultures of normal human keratinocytes (KCs) and N/TERT-1-immortalized KCs, LOX expression was induced during the first differentiation steps. Moreover, the silencing of LOX by stable RNA interference disrupted the expression of early differentiation markers. In a reconstructed-epidermis model, LOX silencing did not impair the stratification process nor the formation of the first differentiated layers. However, terminal differentiation was strongly impaired, as shown by a decreased expression of late differentiation proteins and by the absence of stratum corneum. Nonetheless, inhibition of LOX enzymatic activity by β-aminopropionitrile did not affect the differentiation process. Therefore, LOX protein acts during the first steps of KC differentiation and is important for subsequent commitment into terminal differentiation. Taken together, these results suggest that a finely regulated expression of LOX is necessary for normal KC differentiation and thus for maintenance of epidermal homeostasis

Generation of a conditional Flpo/FRT mouse model expressing constitutively active TGFβ in fibroblasts

International audienceTransforming growth factor (TGFβ) is a secreted factor, which accumulates in tissues during many physio- and pathological processes such as embryonic development, wound healing, fibrosis and cancer. In order to analyze the effects of increased microenvironmental TGFβ concentration in vivo, we developed a conditional transgenic mouse model (Flpo/Frt system) expressing bioactive TGFβ in fibroblasts, a cell population present in the microenvironment of almost all tissues. To achieve this, we created the genetically-engineered [Fsp1-Flpo; FSFTGFβCA] mouse model. The Fsp1-Flpo allele consists in the Flpo recombinase under the control of the Fsp1 (fibroblast-specific promoter 1) promoter. The FSFTGFβCA allele consists in a transgene encoding a constitutively active mutant form of TGFβ (TGFβCA) under the control of a Frt-STOP-Frt (FSF) cassette. The FSFTGFβCA allele was created to generate this model, and functionally validated by in vitro, ex vivo and in vivo techniques. [Fsp1-Flpo; FSFTGFβCA] animals do not present any obvious phenotype despite the correct expression of TGFβCA transgene in fibroblasts. This [Fsp1-Flpo; FSFTGFβCA] model is highly pertinent for future studies on the effect of increased microenvironmental bioactive TGFβ concentrations in mice bearing Cre-dependent genetic alterations in other compartments (epithelial or immune compartments for instance). These dual recombinase system (DRS) approaches will enable scientists to study uncoupled spatiotemporal regulation of different genetic alterations within the same mouse, thus better replicating the complexity of human diseases

TIF1 Suppresses Tumor Progression by Regulating Mitotic Checkpoints and Chromosomal Stability

The transcription accessory factor TIF1γ/TRIM33/RFG7/PTC7/Ectodermin functions as a tumor suppressor that promotes development and cellular differentiation. However, its precise function in cancer has been elusive. In the present study, we report that TIF1γ inactivation causes cells to accumulate chromosomal defects, a hallmark of cancer, due to attenuations in the spindle assembly checkpoint and the post-mitotic checkpoint. TIF1γ deficiency also caused a loss of contact growth inhibition and increased anchorage-independent growth in vitro and in vivo. Clinically, reduced TIF1γ expression in human tumors correlated with an increased rate of genomic rearrangements. Overall, our work indicates that TIF1γ exerts its tumor-suppressive functions in part by promoting chromosomal stability

The TLR3 L412F polymorphism prevents TLR3-mediated tumor cell death induction in pediatric sarcomas

Abstract Toll-like receptor 3 (TLR3) is a pattern recognition receptor mainly known for its role in innate immune response to infection. Indeed, binding of double-stranded RNA (dsRNA) to TLR3 triggers a pro-inflammatory cascade leading to cytokine release and immune cell activation. Its anti-tumoral potential has emerged progressively, associated with a direct impact on tumor cell death induction and with an indirect action on immune system reactivation. Accordingly, TLR3 agonists are currently being tested in clinical trials for several adult cancers. Meanwhile, TLR3 variants have been linked to auto-immune disorders, and as risk factors of viral infection and cancers. However, aside from neuroblastoma, TLR3 role in childhood cancers has not been evaluated. Here, by integrating public transcriptomic data of pediatric tumors, we unveil that high TLR3 expression is largely associated with a better prognosis in childhood sarcomas. Using osteosarcomas and rhabdomyosarcomas as models, we show that TLR3 efficiently drives tumor cell death in vitro and induces tumor regression in vivo. Interestingly, this anti-tumoral effect was lost in cells expressing the homozygous TLR3 L412F polymorphism, which is enriched in a rhabdomyosarcomas cohort. Thus, our results demonstrate the therapeutic potential associated with the targeting of TLR3 in pediatric sarcomas, but also the need to stratify patients eligible for this clinical approach with respect to the TLR3 variants expressed

Tif1γ Suppresses Murine Pancreatic Tumoral Transformation by a Smad4-Independent Pathway

International audienceTranscriptional intermediary factor 1␥ (TIF1␥; alias, TRIM33/RFG7/PTC7/ectodermin) belongs to an evolutionarily conserved family of nuclear factors that have been implicated in stem cell pluripo-tency, embryonic development, and tumor suppression. TIF1␥ expression is markedly down-regulated in human pancreatic tumors, and Pdx1-driven Tif1␥ inactivation cooperates with the Kras G12D onco-gene in the mouse pancreas to induce intraductal papillary mucinous neoplasms. In this study, we report that aged Pdx1-Cre; LSL-Kras G12D ; Tif1␥ lox/lox mice develop pancreatic ductal adenocarcinomas (PDACs), an aggressive and always fatal neoplasm, demonstrating a Tif1␥ tumor-suppressive function in the development of pancreatic carcinogenesis. Deletion of SMAD4/DPC4 (deleted in pancreatic car-cinoma locus 4) occurs in approximately 50% of human cases of PDAC. We, therefore, assessed the genetic relationship between Tif1␥ and Smad4 sig-naling in pancreatic tumors and found that Pdx1-Cre; LSL-Kras G12D ; Smad4 lox/lox ; Tif1␥ lox/lox (alias, KSSTT) mutant mice exhibit accelerated tumor progression. Consequently, Tif1␥ tumor-suppressor effects during progression from a premalignant to a malignant state in our mouse model of pancreatic cancer are independent of Smad4. These findings establish, for the first time to our knowledge, that Tif1␥ and Smad4 both regulate an intraductal papillary mucinous neoplasm-to-PDAC sequence through distinct tumor-suppressor programs

Acinar-to-Ductal Metaplasia Induced by Transforming Growth Factor Beta Facilitates KRASG12D-driven Pancreatic TumorigenesisSummary

Background & Aims: Transforming growth factor beta (TGFβ) acts either as a tumor suppressor or as an oncogene, depending on the cellular context and time of activation. TGFβ activates the canonical SMAD pathway through its interaction with the serine/threonine kinase type I and II heterotetrameric receptors. Previous studies investigating TGFβ-mediated signaling in the pancreas relied either on loss-of-function approaches or on ligand overexpression, and its effects on acinar cells have so far remained elusive. Methods: We developed a transgenic mouse model allowing tamoxifen-inducible and Cre-mediated conditional activation of a constitutively active type I TGFβ receptor (TβRICA) in the pancreatic acinar compartment. Results: We observed that TβRICA expression induced acinar-to-ductal metaplasia (ADM) reprogramming, eventually facilitating the onset of KRASG12D-induced pre-cancerous pancreatic intraepithelial neoplasia. This phenotype was characterized by the cellular activation of apoptosis and dedifferentiation, two hallmarks of ADM, whereas at the molecular level, we evidenced a modulation in the expression of transcription factors such as Hnf1β, Sox9, and Hes1. Conclusions: We demonstrate that TGFβ pathway activation plays a crucial role in pancreatic tumor initiation through its capacity to induce ADM, providing a favorable environment for KRASG12D-dependent carcinogenesis. Such findings are highly relevant for the development of early detection markers and of potentially novel treatments for pancreatic cancer patients. Keywords: Pancreas, Cancer, TGFβ, Acinar-to-Ductal Metaplasia, KRASG12