The genetic programs directing CD4 or CD8 T cell differentiation in the thymus remain poorly understood. While analyzing gene expression during intrathymic T cell selection, we found that Zfp67, encoding the zinc finger transcription factor cKrox, was upregulated during the differentiation of CD4(+) but not CD8(+) T cells. Expression of a cKrox transgene impaired CD8 T cell development and caused major histocompatibility complex class I-restricted thymocytes to differentiate into CD4(+) T cells with helper properties rather than into cytotoxic CD8(+) T cells, as normally found. CD4 lineage differentiation mediated by cKrox required its N-terminal BTB (bric-a-brac, tramtrack, broad complex) domain. These findings identify cKrox as a chief CD4 differentiation factor during positive selection.

AbtractEpithelial–mesenchymal transition (EMT) is an important contributor to the invasion and metastasis of epithelial-derived cancers. While considerable effort has focused in the regulators involved in the transition process, we have focused on consequences of EMT to prosurvival signaling. Changes in distinct metastable and ‘epigentically-fixed’ EMT states were measured by correlation of protein, phosphoprotein, phosphopeptide and RNA transcript abundance. The assembly of 1167 modulated components into functional systems or machines simplified biological understanding and increased prediction confidence highlighting four functional groups: cell adhesion and migration, metabolism, transcription nodes and proliferation/survival networks. A coordinate metabolic reduction in a cluster of 17 free-radical stress pathway components was observed and correlated with reduced glycolytic and increased oxidative phosphorylation enzyme capacity, consistent with reduced cell cycling and reduced need for macromolecular biosynthesis in the mesenchymal state. An attenuation of EGFR autophosphorylation and a switch from autocrine to paracrine-competent EGFR signaling was implicated in the enablement of tumor cell chemotaxis. A similar attenuation of IGF1R, MET and RON signaling with EMT was observed. In contrast, EMT increased prosurvival autocrine IL11/IL6-JAK2-STAT signaling, autocrine fibronectin-integrin α5β1 activation, autocrine Axl/Tyro3/PDGFR/FGFR RTK signaling and autocrine TGFβR signaling. A relatively uniform loss of polarity and cell–cell junction linkages to actin cytoskeleton and intermediate filaments was measured at a systems level. A more heterogeneous gain of ECM remodeling and associated with invasion and migration was observed. Correlation to stem cell, EMT, invasion and metastasis datasets revealed the greatest similarity with normal and cancerous breast stem cell populations, CD49fhi/EpCAM-/lo and CD44hi/CD24lo, respectively.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-010-9367-3) contains supplementary material, which is available to authorized users.

In multiple sclerosis, in which brain tissue becomes permeable to blood proteins, extravascular fibrin deposition correlates with sites of inflammatory demyelination and axonal damage. To examine the role of fibrin in neuroinflammatory demyelination, we depleted fibrin in two tumor necrosis factor transgenic mouse models of multiple sclerosis, transgenic lines TgK21 and Tg6074. In a genetic analysis, we crossed TgK21 mice into a fibrin-deficient background. TgK21fib ؊/؊ mice had decreased inflammation and expression of major histocompatibility complex class I antigens, reduced demyelination, and a lengthened lifespan compared with TgK21 mice. In a pharmacologic analysis, fibrin depletion, by using the snake venom ancrod, in Tg6074 mice also delayed the onset of inflammatory demyelination. Overall, these results indicate that fibrin regulates the inflammatory response in neuroinflammatory diseases. Design of therapeutic strategies based on fibrin depletion could potentially benefit the clinical course of demyelinating diseases such as multiple sclerosis.autoimmunity ͉ anticoagulants ͉ ancrod ͉ extracellular matrix F ibrin, as the final product of the coagulation cascade, plays a major role in blood clotting. However, the role of fibrin is not restricted to the blood, since components of the coagulation cascade reside within tissues and can stimulate extravascular fibrin formation (1). Studies of fibrin deposition in human diseases (2-5), in combination with experiments from genetargeted mice deficient in fibrin (6), have shown that a wide range of pathological conditions, such as glomerulonephritis, lung ischemia, and rheumatoid arthritis, are exacerbated by fibrin deposition.Compromised vasculature in the nervous tissue is a pathogenic manifestation apparent in traumatic injuries, such as spinal cord, optic nerve, and sciatic nerve injury, as well as in central nervous system (CNS) diseases with autoimmune characteristics, such as multiple sclerosis (MS) (7). Blood-brain barrier (BBB) disruption precedes clinical symptoms in MS patients (8), and fibrin is deposited in the lesions (9, 10), apparently before cerebral tissue injury and demyelination (11). Fibrin deposition also coincides with areas of demyelination (12), as well as with areas of axonal damage (13). In addition, in experimental autoimmune encephalomyelitis (EAE), an autoimmune animal model of MS, there is increased coagulation activation before symptom development (14). Pharmacologic depletion of fibrin in EAE ameliorates clinical symptoms, suggesting that fibrin plays a role in CNS inflammatory demyelination (15, 16). Furthermore, inhibition of fibrin formation by attenuation of thrombin activity is protective in the injured optic nerve (17).Although extravascular fibrin(ogen) is present at sites of inflammatory demyelination in MS, and experiments in rodents have established a deleterious role for fibrin in nervous system pathogenesis (18), the cellular mechanisms of fibrin action in the CNS have not been investigated. In this study we examined th...

The progression of cancer from non-metastatic to metastatic is the critical transition in the course of the disease. The epithelial to mesenchymal transition (EMT) is a mechanism by which tumor cells acquire characteristics that improve metastatic efficiency. Targeting EMT processes in patients is therefore a potential strategy to block the transition to metastatic cancer and improve patient outcome. To develop models of EMT applicable to in vitro and in vivo settings, we engineered NCI-H358 non-small cell lung carcinoma cells to inducibly express three well-established drivers of EMT: activated transforming growth factor β (aTGFβ), Snail or Zeb1. We characterized the morphological, molecular and phenotypic changes induced by each of the drivers and compared the different end-states of EMT between the models. Both in vitro and in vivo, induction of the transgenes Snail and Zeb1 resulted in downregulation of epithelial markers and upregulation of mesenchymal markers, and reduced the ability of the cells to proliferate. Induced autocrine expression of aTGFβ caused marker and phenotypic changes consistent with EMT, a modest effect on growth rate, and a shift to a more invasive phenotype. In vivo, this manifested as tumor cell infiltration of the surrounding mouse stromal tissue. Overall, Snail and Zeb1 were sufficient to induce EMT in the cells, but aTGFβ induced a more complex EMT, in which changes in extracellular matrix remodeling components were pronounced.

Prior alcohol use increases vulnerability to cocaine addiction by promoting degradation of HDAC4 and HDAC5.

Epithelial to mesenchymal transition (EMT) plays a dual role in tumor progression. It enhances metastasis of tumor cells by increasing invasive capacity and promoting survival, and it decreases tumor cell sensitivity to epithelial cell-targeting agents such as epithelial growth factor receptor kinase inhibitors. In order to study EMT in tumor cells, we have characterized 3 new models of ligand-driven EMT: the CFPAC1 pancreatic tumor model and the H358 and H1650 lung tumor models. We identified a diverse set of ligands that drives EMT in these models. Hepatocyte growth factor and oncostatin M induced EMT in all models, while transforming growth factor-β induced EMT in both lung models. We observed morphologic, marker and phenotypic changes in response to chronic ligand treatment. Interestingly, stimulation with 2 ligands resulted in more pronounced EMT compared with single-ligand treatment, demonstrating a spectrum of EMT states induced by parallel signaling, such as the JAK and PI3K pathways. The EMT changes observed in response to the ligand were reversed upon ligand withdrawal, demonstrating the ‘metastable’ nature of these models. To study the impact of EMT on cell morphology and invasion in a 3D setting, we cultured cells in a semisolid basement membrane extract. Upon stimulation with EMT ligands, the colonies exhibited changes to EMT markers and showed phenotypes ranging from modest differences in colony architecture (CFPAC1) to complex branching structures (H358, H1650). Collectively, these 3 models offer robust cell systems with which to study the roles that EMT plays in cancer progression.

Resistance to targeted therapies is emerging as a major theme in cancer research. As more therapies become used routinely in the clinic it is apparent that although significant responses are observed the majority of patients progress while on therapy. Drug resistance can occur through a number of distinct mechanisms and no single mechanism can account for all the resistance that occurs in response to a particular therapy. These mechanisms include acquisition of secondary drug-resistant mutations within the target and activation of alternate prosurvival signaling pathways through compensatory signaling or epithelial to mesenchymal transitions (EMT). The ability of cancer cells to undergo an EMT has been implicated as a major factor driving metastasis, through the acquisition of enhanced migratory and invasive properties. However it is also clear that by undergoing this process the cancer cells become resistant to a number of targeted therapies. Recent retrospective analysis of phase 3 clinical trial samples has revealed that a poorer response to Erlotinib in the 2/3rd line setting in NSCLC was associated with a loss of E-cadherin (an epithelial tumor marker), suggesting that tumors that had undergone EMT were less responsive to EGFR-directed therapy. In addition an EMT phenotype has been reported in a number of EGFR-mutant NSCLC patients who have progressed while on erlotinib therapy. These clinical observations suggest that EMT plays an important role in mediating response to targeted therapy. In order to understand the full impact of these clinical observations and identify mechanisms of resistance in mesenchymal tumor cells we have modeled EMT in a number of different ways in vitro. We have used panels of NSCLC cell lines that are in a fixed epithelial or mesenchymal state, induced an EMT with TGFβ, or driven an EMT through prolonged exposure to EGFR-TKi targeted therapy (erlotinib). Using large-scale phosphoproteomic and transcriptomic datasets we used a systems biology approach to uncover important observations relating to the role of EMT as a drug-resistance mechanism. Firstly, these models confirm the clinical observations and show that tumor cells that have undergone EMT are less responsive to a number of targeted agents including EGFR and IGF1R-IR directed agents. Secondly, they reveal the plasticity of the EMT process where three distinct stages of EMT: epithelial, ‘metastable’ mesenchymal and ‘epigenetically-fixed’ mesenchymal are observed. Thirdly, upon undergoing EMT tumor cells acquire novel mechanisms of cellular signaling not apparent in their epithelial counterparts. These include receptor tyrosine kinase (RTK) autocrine and paracrine loops, such as PDGFR, FGFR, AXL and integrin α5β1 and up regulation of IL-6 and IL-11 mediated JAK-STAT signaling. Reciprocal activation of PDGFR signaling through EGFR inhibition was observed in the mesenchymal state. Lastly, these models indicate that as part of the EMT process the tumor cells display a CD44high/CD24low cancer stem cell phenotype and show enhanced colony formation. These observations reinforce the important role that EMT can have in driving drug resistance in tumor cells and highlight the wide diversity of mechanisms that can be used by tumor cells to evade targeted drug therapy. An understanding of these mechanisms and the contexts in which they are most likely to arise will have important implications in driving combinatorial drug therapy in cancer patients in the future.

The EGFR kinase inhibitor erlotinib is approved as a maintenance therapy in 1st line NSCLC as well as for treatment of 2nd/3rd line NSCLC and in combination with Gemcitabine for pancreatic cancer. It has been observed that the most pronounced responses to EGFR tyrosine kinase inhibitors (TKI's) were observed in patients whose tumors expressed a mutated form of the EGFR kinase. These mutations mapped to the kinase domain of the receptor and functionally have been shown to render tumors and cells lines onco-addicted to EGFR signaling. Although patients expressing a mutated EGFR show a dramatic initial response to EGFR TKI's, ∼50% of these patients will progress while on therapy after 1-2 years. The mechanisms that underlie this acquired resistance to EGFR TKI therapy have been intensively studied and include but are not limited to the presence of a second mutation, T790M, or increased HGF-MET signaling. Previously, the role of epithelial to mesenchymal transition (EMT) in resistance to EGFR kinase inhibitors has been described in the context of wild type EGFR. EMT. We were therefore interested in understanding whether EMT could play a role in resistance to EGFR TKIs in the context of an EGFR mutation. Here we show that a panel of NSCLC cell lines, expressing mutant EGFR, can undergo an EMT in response to TGFβ treatment. The cell lines take on a scattered and spindle-like morphology and also down regulate the expression of E-cadherin and up regulate expression of vimentin, classic protein markers of an EMT. Importantly we show that after undergoing EMT, the EGFR mutant line HCC827 has reduced sensitivity to erlotnib treatment which is regained after reversal of the EMT. To further explore whether EMT could play a role in acquired resistance to erlotinib, we generated in vitro cell line models that were resistant to EGFR inhibition through continued culturing in the presence of erlotinib over a 6 month period. Resistant clones generated from parental HCC4006 cells acquired a more scattered and spindle-like morphology consistent with an EMT. These clones had down-regulated E-cadherin and ErbB3 expression and upregulated vimentin, fibronectin and Zeb1 expression and also showed a gene expression pattern consistent with having undergone an EMT. In addition, the resistant H4006 clones were more migratory and invasive than their parental counterpart. Finally we show that the resistant clones are enriched for stem cell markers and have enhanced signaling through the Src family kinases and the JAK-STAT pathway suggesting a mechanistic rationale for their reduced sensitivity to EGFR inhibitors. Taken together. these data indicate that NSCLC cell lines that express a mutant version of EGFR are able to undergo an EMT which can influence the efficacy of EGFR TKIs, suggesting that this may be an additional mechanism underlying the acquired resistance of NSCLC patients to EGFR therapy in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3370. doi:10.1158/1538-7445.AM2011-3370