Prior alcohol use enhances vulnerability to compulsive cocaine self-administration by promoting degradation of HDAC4 and HDAC5.

Addiction to cocaine is commonly preceded by experiences with legal or decriminalized drugs, such as alcohol, nicotine, and marijuana. The biological mechanisms by which these gateway drugs contribute to cocaine addiction are only beginning to be understood. We report that in the rat, prior alcohol consumption results in enhanced addiction-like behavior to cocaine, including continued cocaine use despite aversive consequences. Conversely, prior cocaine use has no effect on alcohol preference. Long-term, but not short-term, alcohol consumption promotes proteasome-mediated degradation of the nuclear histone deacetylases HDAC4 and HDAC5 in the nucleus accumbens, a brain region critical for reward-based memory. Decreased nuclear HDAC activity results in global H3 acetylation, creating a permissive environment for cocaine-induced gene expression. We also find that selective degradation of HDAC4 and HDAC5, facilitated by the class II-specific HDAC inhibitor MC1568, enhances compulsive cocaine self-administration. These results parallel our previously reported findings that the gateway drug nicotine enhances the behavioral effects of cocaine via HDAC inhibition. Together, our findings suggest a shared mechanism of action for the gateway drugs alcohol and nicotine, and reveal a novel mechanism by which environmental factors may alter the epigenetic landscape of the reward system to increase vulnerability to cocaine addiction.

The evaluation of E-Cadherin and vimentin as biomarkers of clinical outcomes among patients with non-small cell lung cancer treated with erlotinib as second- or third-line therapy.

E-Cadherin and vimentin protein expression was assessed in late stage non-small cell lung cancer tumors from the placebo controlled clinical trial, NCIC-CTG BR.21, to determine if these markers had the potential to predict outcome of erlotinib therapy. E-Cadherin and vimentin protein expression levels were assessed in tumors from 95 patients, who were representative of the overall population, using semi-quantitative immunohistochemistry. The percentage of tumor cells with grades 0, 1, 2, or 3 membrane staining of E-cadherin and cytoplasmic staining of vimentin was measured. Three scoring methods and multiple cut-offs were explored to determine if these markers were able to divide patients into groups with different overall survival (OS). A cut-off point for E-cadherin of ≥40% tumor cells with staining of +2 and +3 and a cut-off for vimentin of ≥10% of tumors cell with any staining provided the optimal stratification. The OS hazard ratio (HR) for E-cadherin(+) versus E-cadherin(-) in the erlotinib-treated patients was 0.68 (0.35-1.33) compared with 1.48 (0.69-3.15) in the placebo patients and the OS (HR) for erlotinib versus placebo was 0.47 (0.26-0.88) in E-cadherin(+) patients compared with 1.12 (0.52-2.44) in the E-cadherin(-) patients. The OS (HR) for vimentin(+) versus vimentin(-) in the erlotinib-treated patients was 0.65 (0.31-1.38) compared to 2.32 (1.09-4.94) in the placebo-treated patients and the OS (HR) for erlotinib versus placebo was 0.26 (0.11-0.63) in vimentin(+) compared to 0.99 (0.55-1.76) in the vimentin(-) patients. Similar trends were observed for progression-free survival and response rate. E-Cadherin and vimentin are biomarkers worthy of additional study as predictive markers of outcome of erlotinib therapy.

Inducible expression of TGFß, snail and Zeb1 recapitulates EMT in vitro and in vivo in a NSCLC model.

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 ß (aTGFß), 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 aTGFß 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 aTGFß induced a more complex EMT, in which changes in extracellular matrix remodeling components were pronounced.

A systems view of epithelial-mesenchymal transition signaling states.

Epithelial-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 α5ß1 activation, autocrine Axl/Tyro3/PDGFR/FGFR RTK signaling and autocrine TGFßR 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, CD49f(hi)/EpCAM(-/lo) and CD44(hi)/CD24(lo), respectively.

Cooperative signaling between oncostatin M, hepatocyte growth factor and transforming growth factor-ß enhances epithelial to mesenchymal transition in lung and pancreatic tumor models.

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-ß 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.

The zinc finger protein cKrox directs CD4 lineage differentiation during intrathymic T cell positive selection.

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.

Fibrin depletion decreases inflammation and delays the onset of demyelination in a tumor necrosis factor transgenic mouse model for multiple sclerosis.

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.