Inactivation of TIF1γ Cooperates with KrasG12D to Induce Cystic Tumors of the Pancreas

Inactivation of the Transforming Growth Factor Beta (TGFβ) tumor suppressor pathway contributes to the progression of Pancreatic Ductal AdenoCarcinoma (PDAC) since it is inactivated in virtually all cases of this malignancy. Genetic lesions inactivating this pathway contribute to pancreatic tumor progression in mouse models. Transcriptional Intermediary Factor 1 gamma (TIF1γ) has recently been proposed to be involved in TGFβ signaling, functioning as either a positive or negative regulator of the pathway. Here, we addressed the role of TIF1γ in pancreatic carcinogenesis. Using conditional Tif1γ knockout mice (Tif1γlox/lox), we selectively abrogated Tif1γ expression in the pancreas of Pdx1-Cre;Tif1γlox/lox mice. We also generated Pdx1-Cre;LSL-KrasG12D;Tif1γlox/lox mice to address the effect of Tif1γ loss-of-function in precancerous lesions induced by oncogenic KrasG12D. Finally, we analyzed TIF1γ expression in human pancreatic tumors. In our mouse model, we showed that Tif1γ was dispensable for normal pancreatic development but cooperated with Kras activation to induce pancreatic tumors reminiscent of human Intraductal Papillary Mucinous Neoplasms (IPMNs). Interestingly, these cystic lesions resemble those observed in Pdx1-Cre;LSL-KrasG12D;Smad4lox/lox mice described by others. However, distinctive characteristics, such as the systematic presence of endocrine pseudo-islets within the papillary projections, suggest that SMAD4 and TIF1γ don't have strictly redundant functions. Finally, we report that TIF1γ expression is markedly down-regulated in human pancreatic tumors by quantitative RT–PCR and immunohistochemistry supporting the relevance of these findings to human malignancy. This study suggests that TIF1γ is critical for tumor suppression in the pancreas, brings new insight into the genetics of pancreatic cancer, and constitutes a promising model to decipher the respective roles of SMAD4 and TIF1γ in the multifaceted functions of TGFβ in carcinogenesis and development

Caractérisation des étapes précoces de l'infection par le HIV-1 dans les monocytes, les macrophages et les lymphocytes

Le Virus de l Immunodéficience Humaine de type 1 (HIV-1) cible principalement les lymphocytes T, les monocytes, les macrophages et les cellules dendritiques. L'infection de ces cellules débute par une série d événements définis comme les étapes précoces du cycle viral. Notre objectif était de caractériser les effets des facteurs clés viraux et cellulaires intervenant lors de ces étapes, et d étudier le comportement encore peu caractérisé des génomes viraux infectieux. Nous avons exploré les étapes précoces de l infection dans les cellules cibles du HIV-1 en établissant de manière systématique les cinétiques de décapsidation, de synthèse et d intégration de l ADN viral, ainsi que la cinétique de stabilité fonctionnelle des complexes de transcription inverse (RTC). Contrairement aux précédentes études, nous avons montré que les monocytes supportent l infection par le HIV-1, même si celle-ci est beaucoup plus lente que dans les autres types cellulaires. Pour les types cellulaires plus permissifs, nous avons montré que l infection était caractérisée par des événements spécifiques à chaque type cellulaire. Ainsi, notre analyse a révélé l existence d un délai constant entre les étapes de transcription inverse et d intégration. Ce délai reflète probablement le temps nécessaire à l import nucléaire, et non au trafic des RTC par le cytosquelette qui ne semble pas avoir de rôle majeur lors des étapes précoces de l infection. De plus, nous avons montré que les processus de décapsidation et de transcription inverse s effectuent en parallèle, et non de manière séquentielle comme proposé précédemment. Enfin, de grandes différences existent entre les différents types cellulaires en ce qui concerne la stabilité fonctionnelle des RTC. Toutes ces données suggèrent que la susceptibilité des cellules à l infection résulte d une balance entre vitesse de transcription inverse et stabilité fonctionnelle des RTC, caractéristique de chaque type cellulaire.The Human Immunodeficiency Virus Type I (HIV-1) preferentially targets T cells, monocytes, macrophages and dendritic cells. The infection of these cells begins with a series of events defined as the early steps of the viral life cycle. Our objective was to characterize these steps by characterizing the behaviour of infectious viral genomes that, for reasons yet unknown, constitute only a minor fraction of the total. We explored the early steps of viral infection in HIV-1 target cells by determining systematically the kinetics of viral DNA synthesis, integration, uncoating, as well as by introducing and measuring a novel parameter in the stability of viral complexes at their pre-RT state. In contrast to previous reports, we have shown that non-stimulated monocytes support HIV-1 infection instead of restricting it, although the early steps of infection proceed with extremely long kinetics compared to the other cell types. In cells more permissive to HIV-1 infection, our analysis revealed that infection is characterized by a series of parameters that are specific to a given cell type. Overall, our results indicated the existence of a constant delay between the end of reverse transcription and integration, even in the absence of an intact cytoskeleton. Moreover, we have shown that uncoating proceeds together with but not after reverse transcription. Finally, there are significant differences between the different cell types, in terms of functional stability of RTCs Our comparative analysis indicates that the success of HIV-1 infection may be governed by a cell type specific balance between the speed of reverse transcription and the stability of functional RTCs.LYON-ENS Sciences (693872304) / SudocSudocFranceF

Characterization of the Early Steps of Infection of Primary Blood Monocytes by Human Immunodeficiency Virus Type 1▿

Blood-circulating monocytes migrate in tissues in response to danger stimuli and differentiate there into two major actors of the immune system: macrophages and dendritic cells. Given their migratory behavior and their pivotal role in the orchestration of immune responses, it is not surprising that cells of the monocyte lineage are the target of several viruses, including human immunodeficiency virus type 1 (HIV-1). HIV-1 replicates in monocytoid cells to an extent that is influenced by their differentiation status and modulated by exogenous stimulations. Unstimulated monocytes display a relative resistance to HIV infection mostly exerted during the early steps of the viral life cycle. Despite intensive studies, the identity of the affected step remains controversial, although it is generally assumed to take place after viral entry. We reexamine here the early steps of viral infection of unstimulated monocytes using vesicular stomatitis virus G protein-pseudotyped HIV-1 virions. Our data indicate that a first block to the early steps of infection of monocytes with these particles occurs at the level of viral entry. After entry, reverse transcription and integration proceed with extremely slow kinetics rather than being blocked. Once completed, viral DNA molecules delay entry into the nucleus and integration for up to 5 to 6 days. The inefficacy of these steps accounts for the resistance of monocytes to HIV-1 during the early steps of infection

Characterization of the Behavior of Functional Viral Genomes during the Early Steps of Human Immunodeficiency Virus Type 1 Infection▿

Infectious viral DNA constitutes only a small fraction of the total viral DNA produced during retroviral infection, and as such its exact behavior is largely unknown. In the present study, we characterized in detail functional viral DNA produced during the early steps of human immunodeficiency virus type 1 infection by analyzing systematically their kinetics of synthesis and integration in different target cells. In addition, we have compared the functional stability of viral nucleoprotein complexes arrested at their pre-reverse transcription state, and we have attempted to measure the kinetics of loss of capsid proteins from viral complexes through the susceptibility of the early phases of infection to cyclosporine, a known inhibitor of the interaction between viral capsid and cyclophilin A. Overall, our data suggest a model in which loss of capsid proteins from viral complexes and reverse transcription occur concomitantly and in which the susceptibility of target cells to infection results from a competition between the ability of the cellular environment to quickly destabilize viral nucleoprotein complexes and the capability of the virus to escape such targeting by engaging the reverse transcription reaction

Characterization of Simian Immunodeficiency Virus SIVSM/Human Immunodeficiency Virus Type 2 Vpx Function in Human Myeloid Cells▿

Human immunodeficiency virus type 2 (HIV-2)/simian immunodeficiency virus SIVSM Vpx is incorporated into virion particles and is thus present during the early steps of infection, when it has been reported to influence the nuclear import of viral DNA. We recently reported that Vpx promoted the accumulation of full-length viral DNA following the infection of human monocyte-derived dendritic cells (DCs). This positive effect was exerted following the infection of DCs with cognate viruses and with retroviruses as divergent as HIV-1, feline immunodeficiency virus, and even murine leukemia virus, leading us to suggest that Vpx counteracted an antiviral restriction present in DCs. Here, we show that Vpx is required, albeit to a different extent, for the infection of all myeloid but not of lymphoid cells, including monocytes, macrophages, and monocytoid THP-1 cells that had been induced to differentiate with phorbol esters. The intracellular localization of Vpx was highly heterogeneous and cell type dependent, since Vpx localized differently in HeLa cells and DCs. Despite these differences, no clear correlation between the functionality of Vpx and its intracellular localization could be drawn. As a first insight into its function, we determined that SIVSM/HIV-2 and SIVRCM Vpx proteins interact with the DCAF1 adaptor of the Cul4-based E3 ubiquitin ligase complex recently described to associate with HIV-1 Vpr and HIV-2 Vpx. However, the functionality of Vpx proteins in the infection of DCs did not strictly correlate with DCAF1 binding, and knockdown experiments failed to reveal a functional role for this association in differentiated THP-1 cells. Lastly, when transferred in the context of a replication-competent viral clone, Vpx was required for replication in DCs

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

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