Transient receptor potential vanilloid 4 channel inhibition attenuates lung ischemia-reperfusion injury in a porcine lung transplant model.

OBJECTIVE: Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel important in many physiological and pathophysiological processes, including pulmonary disease. Using a murine model, we previously demonstrated that TRPV4 mediates lung ischemia-reperfusion injury, the major cause of primary graft dysfunction after transplant. The current study tests the hypothesis that treatment with a TRPV4 inhibitor will attenuate lung ischemia-reperfusion injury in a clinically relevant porcine lung transplant model. METHODS: A porcine left-lung transplant model was used. Animals were randomized to 2 treatment groups (n = 5/group): vehicle or GSK2193874 (selective TRPV4 inhibitor). Donor lungs underwent 30 minutes of warm ischemia and 24 hours of cold preservation before left lung allotransplantation and 4 hours of reperfusion. Vehicle or GSK2193874 (1 mg/kg) was administered to the recipient as a systemic infusion after recipient lung explant. Lung function, injury, and inflammatory biomarkers were compared. RESULTS: After transplant, left lung oxygenation was significantly improved in the TRPV4 inhibitor group after 3 and 4 hours of reperfusion. Lung histology scores and edema were significantly improved, and neutrophil infiltration was significantly reduced in the TRPV4 inhibitor group. TRPV4 inhibitor-treated recipients had significantly reduced expression of interleukin-8, high mobility group box 1, P-selectin, and tight junction proteins (occludin, claudin-5, and zonula occludens-1) in bronchoalveolar lavage fluid as well as reduced angiopoietin-2 in plasma, all indicative of preservation of endothelial barrier function. CONCLUSIONS: Treatment of lung transplant recipients with TRPV4 inhibitor significantly improves lung function and attenuates ischemia-reperfusion injury. Thus, selective TRPV4 inhibition may be a promising therapeutic strategy to prevent primary graft dysfunction after transplant.

The endothelium: gatekeeper to lung ischemia-reperfusion injury.

The success of lung transplantation is limited by the high rate of primary graft dysfunction due to ischemia-reperfusion injury (IRI). Lung IRI is characterized by a robust inflammatory response, lung dysfunction, endothelial barrier disruption, oxidative stress, vascular permeability, edema, and neutrophil infiltration. These events are dependent on the health of the endothelium, which is a primary target of IRI that results in pulmonary endothelial barrier dysfunction. Over the past 10 years, research has focused more on the endothelium, which is beginning to unravel the multi-factorial pathogenesis and immunologic mechanisms underlying IRI. Many important proteins, receptors, and signaling pathways that are involved in the pathogenesis of endothelial dysfunction after IR are starting to be identified and targeted as prospective therapies for lung IRI. In this review, we highlight the more significant mediators of IRI-induced endothelial dysfunction discovered over the past decade including the extracellular glycocalyx, endothelial ion channels, purinergic receptors, kinases, and integrins. While there are no definitive clinical therapies currently available to prevent lung IRI, we will discuss potential clinical strategies for targeting the endothelium for the treatment or prevention of IRI. The accruing evidence on the essential role the endothelium plays in lung IRI suggests that promising endothelial-directed treatments may be approaching the clinic soon. The application of therapies targeting the pulmonary endothelium may help to halt this rapid and potentially fatal injury.

Purinergic P2Y2 receptor-induced activation of endothelial TRPV4 channels mediates lung ischemia-reperfusion injury.

Lung ischemia-reperfusion injury (IRI), characterized by inflammation, vascular permeability, and lung edema, is the major cause of primary graft dysfunction after lung transplantation. Here, we investigated the cellular mechanisms underlying lung IR-induced activation of endothelial TRPV4 channels, which play a central role in lung edema and dysfunction after IR. In a left lung hilar-ligation model of IRI in mice, we found that lung IRI increased the efflux of ATP through pannexin 1 (Panx1) channels at the endothelial cell (EC) membrane. Elevated extracellular ATP activated Ca2+ influx through endothelial TRPV4 channels downstream of purinergic P2Y2 receptor (P2Y2R) signaling. P2Y2R-dependent activation of TRPV4 channels was also observed in human and mouse pulmonary microvascular endothelium in ex vivo and in vitro models of IR. Endothelium-specific deletion of P2Y2R, TRPV4, or Panx1 in mice substantially prevented lung IRI-induced activation of endothelial TRPV4 channels and lung edema, inflammation, and dysfunction. These results identify endothelial P2Y2R as a mediator of the pathological sequelae of IRI in the lung and show that disruption of the endothelial Panx1-P2Y2R-TRPV4 signaling pathway could be a promising therapeutic strategy for preventing lung IRI after transplantation.

Steen solution protects pulmonary microvascular endothelial cells and preserves endothelial barrier after lipopolysaccharide-induced injury.

OBJECTIVES: Acute respiratory distress syndrome represents the devastating result of acute lung injury, with high mortality. Limited methods are available for rehabilitation of lungs affected by acute respiratory distress syndrome. Our laboratory has demonstrated rehabilitation of sepsis-injured lungs via normothermic ex vivo and in vivo perfusion with Steen solution (Steen). However, mechanisms responsible for the protective effects of Steen remain unclear. This study tests the hypothesis that Steen directly attenuates pulmonary endothelial barrier dysfunction and inflammation induced by lipopolysaccharide. METHODS: Primary pulmonary microvascular endothelial cells were exposed to lipopolysaccharide for 4 hours and then recovered for 8 hours in complete media (Media), Steen, or Steen followed by complete media (Steen/Media). Oxidative stress, chemokines, permeability, interendothelial junction proteins, and toll-like receptor 4-mediated pathways were assessed in pulmonary microvascular endothelial cells using standard methods. RESULTS: Lipopolysaccharide treatment of pulmonary microvascular endothelial cells and recovery in Media significantly induced reactive oxygen species, lipid peroxidation, expression of chemokines (eg, chemokine [C-X-C motif] ligand 1 and C-C motif chemokine ligand 2) and cell adhesion molecules (P-selectin, E-selectin, and vascular cell adhesion molecule 1), permeability, neutrophil transmigration, p38 mitogen-activated protein kinase and nuclear factor kappa B signaling, and decreased expression of tight and adherens junction proteins (zonula occludens-1, zonula occludens-2, and vascular endothelial-cadherin). All of these inflammatory pathways were significantly attenuated after recovery of pulmonary microvascular endothelial cells in Steen or Steen/Media. CONCLUSIONS: Steen solution preserves pulmonary endothelial barrier function after lipopolysaccharide exposure by promoting an anti-inflammatory environment via attenuation of oxidative stress, toll-like receptor 4-mediated signaling, and conservation of interendothelial junctions. These protective mechanisms offer insight into the advancement of methods for in vivo lung perfusion with Steen for the treatment of severe acute respiratory distress syndrome.

Gastric Aspiration and Ventilator-Induced Model of Acute Respiratory Distress Syndrome in Swine.

INTRODUCTION: Mainstays of current treatment for acute respiratory distress syndrome (ARDS) focus on supportive care and rely on intrinsic organ recovery. Animal models of ARDS are often limited by systemic injury. We hypothesize that superimposing gastric aspiration and ventilator-induced injury will induce a lung-specific injury model of severe ARDS. MATERIALS AND METHODS: Adult swine (n = 8) were subject to a 12 h injury development period followed by 24 h of post-injury monitoring. Lung injury was induced with gastric secretions (3 cc/kg body weight/lung, pH 1-2) instilled to bilateral mainstem bronchi under direct bronchoscopic vision. Ventilator settings within the injury period contradicted baseline settings using high tidal volumes and low positive end-expiratory pressure. Baseline settings were restored following the injury period. Arterial oxygenation and lung compliance were monitored. RESULTS: At 12 h, PaO2/FiO2 ratio and static and dynamic compliance were significantly reduced from baseline (P < 0.05). During the postinjury period, animals showed no signs of recovery in PaO2/FiO2 ratio and lung compliance. Lung edema (wet/dry weight ratio) of injured lungs was significantly elevated versus noninjured lungs (8.5 ± 1.7 versus 5.6 ± 0.3, P = 0.009). Expression of proinflammatory cytokines IL-6 and IL-8 were significantly elevated in injured lungs (P < 0.05). CONCLUSIONS: Twelve hours of high tidal volume and low positive end-expiratory pressure in conjunction with low-pH gastric content instillation produces significant acute lung injury in swine. This large animal model may be useful for testing severe ARDS treatment strategies.

Two Hours of In Vivo Lung Perfusion Improves Lung Function in Sepsis-Induced Acute Respiratory Distress Syndrome.

Sepsis is the leading cause of acute respiratory distress syndrome (ARDS) in adults and carries a high mortality. Utilizing a previously validated porcine model of sepsis-induced ARDS, we sought to refine our novel therapeutic technique of in vivo lung perfusion (IVLP). We hypothesized that 2 hours of IVLP would provide non-inferior lung rehabilitation compared to 4 hours of treatment. Adult swine (n = 8) received lipopolysaccharide to develop ARDS and were placed on central venoarterial extracorporeal membrane oxygenation. Animals were randomized to 2 vs 4 hours of IVLP. The left pulmonary vessels were cannulated to IVLP using antegrade Steen solution. After IVLP treatment, the left lung was decannulated and reperfused for 4 hours. Total lung compliance and pulmonary venous gases from the right lung (control) and left lung (treatment) were sampled hourly. Biochemical analysis of tissue and bronchioalveolar lavage was performed along with tissue histologic assessment. Throughout IVLP and reperfusion, treated left lung PaO2/FiO2 ratio was significantly higher than the right lung control in the 2-hour group (332.2 ± 58.9 vs 264.4 ± 46.5, P = 0.01). In the 4-hour group, there was no difference between treatment and control lung PaO2/FiO2 ratio (258.5 ± 72.4 vs 253.2 ± 90.3, P = 0.58). Wet-to-dry weight ratios demonstrated reduced edema in the treated left lungs of the 2-hour group (6.23 ± 0.73 vs 7.28 ± 0.61, P = 0.03). Total lung compliance was also significantly improved in the 2-hour group. Two hours of IVLP demonstrated superior lung function in this preclinical model of sepsis-induced ARDS. Clinical translation of IVLP may shorten duration of mechanical support and improve outcomes.

Endothelial Transient Receptor Potential Vanilloid 4 Channels Mediate Lung Ischemia-Reperfusion Injury.

BACKGROUND: Lung ischemia-reperfusion injury (IRI), involving severe inflammation and edema, is a major cause of primary graft dysfunction after transplant. Activation of transient receptor potential vanilloid 4 (TRPV4) channels modulates vascular permeability. Thus, this study tests the hypothesis that endothelial TRPV4 channels mediate lung IRI. METHODS: A left lung hilar-ligation model was used to induce lung IR in C57BL/6 wild-type (WT), Trpv4-/-, tamoxifen-inducible endothelial Trpv4 knockout (Trpv4EC-/-), and tamoxifen-treated control (Trpv4fl/fl) (n ≥ 6 mice/group). WT mice were also treated with GSK2193874 (WT+GSK219), a TRPV4-specific inhibitor (1 mg/kg). Partial pressure of arterial oxygen, edema (wet-to-dry weight ratio), compliance, neutrophil infiltration, and cytokine concentrations in bronchoalveolar lavage fluid were assessed. Pulmonary microvascular endothelial cells were characterized in vitro after exposure to hypoxia-reoxygenation. RESULTS: Compared with WT, partial pressure of arterial oxygen after IR was significantly improved in Trpv4-/- mice (133.1 ± 43.9 vs 427.8 ± 83.1 mm Hg, P < .001) and WT+GSK219 mice (133.1 ± 43.9 vs 447.0 ± 67.6 mm Hg, P < .001). Pulmonary edema and neutrophil infiltration were also significantly reduced after IR in Trpv4-/- and WT+GSK219 mice vs WT. Trpv4EC-/- mice after IR demonstrated significantly improved oxygenation vs control (109.2 ± 21.6 vs 405.3 ± 41.4 mm Hg, P < .001) as well as significantly improved compliance and significantly less edema, neutrophil infiltration, and proinflammatory cytokine production (tumor necrosis factor-a, chemokine [C-X-C motif] ligand 1, interleukin 17, interferon-γ). Hypoxia-reoxygenation-induced permeability and chemokine (C-X-C motif) ligand 1 expression by pulmonary microvascular endothelial cells were significantly attenuated by TRPV4 inhibitors. CONCLUSIONS: Endothelial TRPV4 plays a key role in vascular permeability and lung inflammation after IR. TRPV4 channels may be a promising therapeutic target to mitigate lung IRI and decrease the incidence of primary graft dysfunction after transplant.

Secondary Burn Progression Mitigated by an Adenosine 2A Receptor Agonist.

Current burn therapy is largely supportive with limited therapies to curb secondary burn progression. Adenosine 2A receptor (A2AR) agonists have anti-inflammatory effects with decreased inflammatory cell infiltrate and release of proinflammatory mediators. Using a porcine comb burn model, we examined whether A2AR agonists could mitigate burn progression. Eight full-thickness comb burns (four prongs with three spaces per comb) per pig were generated with the following specifications: temperature 115°C, 3-kg force, and 30-second application time. In a randomized fashion, animals (four per group) were then treated with A2AR agonist (ATL-1223, 3 ng/kg/min, intravenous infusion over 6 hours) or vehicle control. Necrotic interspace development was the primary outcome and additional histologic assessments were conducted. Analysis of unburned interspaces (72 per group) revealed that ATL-1223 treatment decreased the rate of necrotic interspace development over the first 4 days following injury (p < .05). Treatment significantly decreased dermal neutrophil infiltration at 48 hours following burn (14.63 ± 4.30 vs 29.71 ± 10.76 neutrophils/high-power field, p = .029). Additionally, ATL-1223 treatment was associated with fewer interspaces with evidence of microvascular thrombi through postburn day 4 (18.8% vs 56.3%, p = .002). Two weeks following insult, the depth of injury at distinct burn sites (adjacent to interspaces) was significantly reduced by ATL-1223 treatment (2.91 ± 0.47 vs 3.28 ± 0.58 mm, p = .038). This work demonstrates the ability of an A2AR agonist to mitigate burn progression through dampening local inflammatory processes. Extended dosing strategies may yield additional benefit and improve cosmetic outcome in those with severe injury.

A detailed characterization of stepwise activation of the androgen receptor variant 7 in prostate cancer cells.

Expression of the androgen receptor splice variant 7 (AR-V7) is frequently detected in castrate resistant prostate cancer and associated with resistance to AR-targeted therapies. While we have previously noted that homodimerization is required for the transcriptional activity of AR-V7 and that AR-V7 can also form heterodimers with the full-length AR (AR-FL), there are still many gaps of knowledge in AR-V7 stepwise activation. In the present study, we show that neither AR-V7 homodimerization nor AR-V7/AR-FL heterodimerization requires cofactors or DNA binding. AR-V7 can enter the nucleus as a monomer and drive a transcriptional program and DNA-damage repair as a homodimer. While forming a heterodimer with AR-FL to induce nuclear localization of unliganded AR-FL, AR-V7 does not need to interact with AR-FL to drive gene transcription or DNA-damage repair in prostate cancer cells that co-express AR-V7 and AR-FL. These data indicate that AR-V7 can function independently of its interaction with AR-FL in the true castrate state or "absence of ligand", providing support for the utility of targeting AR-V7 in improving outcomes of patients with castrate resistant prostate cancer.

AR phosphorylation and CHK2 kinase activity regulates IR-stabilized AR-CHK2 interaction and prostate cancer survival.

We have previously demonstrated that checkpoint kinase 2 (CHK2) is a critical negative regulator of androgen receptor (AR) transcriptional activity, prostate cancer (PCa) cell growth, and androgen sensitivity. We have now uncovered that the AR directly interacts with CHK2 and ionizing radiation (IR) increases this interaction. This IR-induced increase in AR-CHK2 interactions requires AR phosphorylation and CHK2 kinase activity. PCa associated CHK2 mutants with impaired kinase activity reduced IR-induced AR-CHK2 interactions. The destabilization of AR - CHK2 interactions induced by CHK2 variants impairs CHK2 negative regulation of cell growth. CHK2 depletion increases transcription of DNAPK and RAD54, increases clonogenic survival, and increases resolution of DNA double strand breaks. The data support a model where CHK2 sequesters the AR through direct binding decreasing AR transcription and suppressing PCa cell growth. CHK2 mutation or loss of expression thereby leads to increased AR transcriptional activity and survival in response to DNA damage.

Discovery of a novel long noncoding RNA overlapping the LCK gene that regulates prostate cancer cell growth.

BACKGROUND: Virtually all patients with metastatic prostate cancer (PCa) will relapse and develop lethal castration-resistant prostate cancer (CRPC). Long noncoding RNAs (lncRNAs) are emerging as critical regulatory elements of many cellular biological processes, and may serve as therapeutic targets for combating PCa progression. Here, we have discovered in a high-throughput RNAi screen a novel lncRNA in PCa, and assessed the oncogenic effects of this lncRNA. METHODS: Rapid amplification of cDNA ends and sequencing was utilized to identify a previously unannotated lncRNA lying within exon six and the 3'UTR of the lymphocyte-specific protein tyrosine kinase (LCK) gene. The levels of HULLK in the presence or absence of hormone and/or enzalutamide or coregulator inhibitors were measured by quantitative PCR (qPCR). The determination of HULLK transcription and localization were characterized by strand-specific qPCR and cellular fractionation followed by qPCR, respectively. The correlation between HULLK expression and prostate cancer Gleason score was analyzed by droplet digital PCR. CyQuant assays were conducted to evaluate the effects of knocking down HULLK with shRNAs or overexpressing HULLK on cell growth. RESULTS: In this study, a previously unannotated lncRNA lying within exon six and 3'UTR of the LCK gene was dramatically upregulated by androgen in a dose-dependent manner, and the anti-androgen enzalutamide completely blocked this hormone-induced increase. Therefore, we labeled this lncRNA "HULLK" for Hormone-Upregulated lncRNA within LCK. Binding sites for two AR coregulators p300 and Brd4 reside near the HULLK transcriptional start site (TSS), and inhibitors of these coregulators downregulated HULLK. HULLK is transcribed from the sense strand of DNA, and predominantly localizes to the cytoplasm. HULLK transcripts are not only expressed in prostate cancer cell lines, but also prostate cancer patient tissue. Remarkably, there was a significant positive correlation between HULLK expression and high-grade PCa in multiple cohorts. shRNAs targeting HULLK significantly decreased PCa cell growth. Moreover, cells overexpressing HULLK were hypersensitive to androgen stimulation. CONCLUSIONS: HULLK is a novel lncRNA situated within the LCK gene that may serve as an oncogene in PCa. Our data enhances our understanding of lncRNA biology and may assist in the development of additional biomarkers or more effective therapeutic targets for advanced PCa.

Checkpoint Kinase 2 Negatively Regulates Androgen Sensitivity and Prostate Cancer Cell Growth.

Prostate cancer is the second leading cause of cancer death in American men, and curing metastatic disease remains a significant challenge. Nearly all patients with disseminated prostate cancer initially respond to androgen deprivation therapy (ADT), but virtually all patients will relapse and develop incurable castration-resistant prostate cancer (CRPC). A high-throughput RNAi screen to identify signaling pathways regulating prostate cancer cell growth led to our discovery that checkpoint kinase 2 (CHK2) knockdown dramatically increased prostate cancer growth and hypersensitized cells to low androgen levels. Mechanistic investigations revealed that the effects of CHK2 were dependent on the downstream signaling proteins CDC25C and CDK1. Moreover, CHK2 depletion increased androgen receptor (AR) transcriptional activity on androgen-regulated genes, substantiating the finding that CHK2 affects prostate cancer proliferation, partly, through the AR. Remarkably, we further show that CHK2 is a novel AR-repressed gene, suggestive of a negative feedback loop between CHK2 and AR. In addition, we provide evidence that CHK2 physically associates with the AR and that cell-cycle inhibition increased this association. Finally, IHC analysis of CHK2 in prostate cancer patient samples demonstrated a decrease in CHK2 expression in high-grade tumors. In conclusion, we propose that CHK2 is a negative regulator of androgen sensitivity and prostate cancer growth, and that CHK2 signaling is lost during prostate cancer progression to castration resistance. Thus, perturbing CHK2 signaling may offer a new therapeutic approach for sensitizing CRPC to ADT and radiation.

The convergence of DNA damage checkpoint pathways and androgen receptor signaling in prostate cancer.

It is increasingly clear that castration-resistant prostate cancer (PCa) is dependent on the androgen receptor (AR). This has led to the use of anti-androgen therapies that reduce endogenous steroid hormone production as well as the use of AR antagonists. However, the AR does not act in isolation and integrates with a milieu of cell-signaling proteins to affect cell biology. It is well established that cancer is a genetic disease resulting from the accumulation of mutations and chromosomal translocations that enables cancer cells to survive, proliferate, and disseminate. To maintain genomic integrity, there exists conserved checkpoint signaling pathways to facilitate cell cycle delay, DNA repair, and/or apoptosis in response to DNA damage. The AR interacts with, affects, and is affected by these DNA damage-response proteins. This review will focus on the connections between checkpoint signaling and the AR in PCa. We will describe what is known about how components of checkpoint signaling regulate AR activity and what questions still face the field.

Androgen receptor phosphorylation: biological context and functional consequences.

The androgen receptor (AR) is a ligand-regulated transcription factor that belongs to the family of nuclear receptors. In addition to regulation by steroid, the AR is also regulated by post-translational modifications generated by signal transduction pathways. Thus, the AR functions not only as a transcription factor but also as a node that integrates multiple extracellular signals. The AR plays an important role in many diseases, including complete androgen insensitivity syndrome, spinal bulbar muscular atrophy, prostate and breast cancer, etc. In the case of prostate cancer, dependence on AR signaling has been exploited for therapeutic intervention for decades. However, the effectiveness of these therapies is limited in advanced disease due to restoration of AR signaling. Greater understanding of the molecular mechanisms involved in AR action will enable the development of improved therapeutics to treat the wide range of AR-dependent diseases. The AR is subject to regulation by a number of kinases through post-translational modifications on serine, threonine, and tyrosine residues. In this paper, we review the AR phosphorylation sites, the kinases responsible for these phosphorylations, as well as the biological context and the functional consequences of these phosphorylations. Finally, what is known about the state of AR phosphorylation in clinical samples is discussed.

An Inv(16)(p13.3q24.3)-encoded CBFA2T3-GLIS2 fusion protein defines an aggressive subtype of pediatric acute megakaryoblastic leukemia.

To define the mutation spectrum in non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL samples. Our analysis identified a cryptic chromosome 16 inversion (inv(16)(p13.3q24.3)) in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.

A novel association between p130Cas and resistance to the chemotherapeutic drug adriamycin in human breast cancer cells.

Resistance to chemotherapy remains a major obstacle for the treatment of breast cancer. Understanding the molecular mechanism(s) of resistance is crucial for the development of new effective therapies to treat this disease. This study examines the putative role of p130(Cas) (Cas) in resistance to the cytotoxic agent Adriamycin. High expression of Cas in primary breast tumors is associated with the failure to respond to the antiestrogen tamoxifen and poor prognosis, highlighting the potential clinical importance of this molecule. Here, we show a novel association between Cas and resistance to Adriamycin. We show that Cas overexpression renders MCF-7 breast cancer cells less sensitive to the growth inhibitory and proapoptotic effects of Adriamycin. The catalytic activity of the nonreceptor tyrosine kinase c-Src, but not the epidermal growth factor receptor, is critical for Cas-mediated protection from Adriamycin-induced death. The phosphorylation of Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) is elevated in Cas-overexpressing cells treated with Adriamycin, whereas expression of the proapoptotic protein Bak is decreased. Conversely, Cas depletion in the more resistant T47D and MDA-MB-231 cell lines increases sensitivity to Adriamycin. Based on these data, we propose that Cas activates growth and survival pathways regulated by c-Src, Akt, and ERK1/2 that lead to the inhibition of mitochondrial-mediated apoptosis in the presence of Adriamycin. Because Cas is frequently expressed at high levels in breast cancers, these findings raise the possibility of resensitizing Cas-overexpressing tumors to chemotherapy through perturbation of Cas signaling pathways.

Physical and functional interactions between Cas and c-Src induce tamoxifen resistance of breast cancer cells through pathways involving epidermal growth factor receptor and signal transducer and activator of transcription 5b.

High expression of the adaptor molecule Cas has been linked to resistance to the antiestrogen tamoxifen, both in tissue culture and in human tumors. The aim of this study was to elucidate the mechanism(s) by which overexpression of Cas confers resistance to tamoxifen. Cas overexpression in MCF-7 breast cancer cells was shown to alleviate both tamoxifen-mediated growth inhibition and induction of apoptosis. This enhancement of cell proliferation/survival occurred in the absence of detectable effects on estrogen receptor (ER) transcriptional activity under conditions where tamoxifen was present, indicating that Cas-dependent tamoxifen resistance is not the result of a switch to an ER-negative phenotype or enhanced responses to the partial agonist activity of tamoxifen. Instead, we present evidence, suggesting that Cas promotes tamoxifen resistance by deregulation of alternative cell proliferation pathways, particularly those mediated through enhanced c-Src protein tyrosine kinase activity arising from Cas/c-Src interactions. Overexpression of Cas was found to drive endogenous c-Src into complex with Cas, a process that has been shown previously to cause up-regulation of c-Src tyrosine kinase activity. MCF-7 cells overexpressing Cas exhibited increased phosphorylation of two c-Src substrates, Tyr845 in the kinase domain of the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription (STAT) 5b. Importantly, Cas-dependent protection from the antiproliferative effects of tamoxifen was reversed by the expression of dominant inhibitory variants of these substrates (Y845F EGFR and COOH-terminally truncated STAT5b). Based on these findings, we suggest that the Cas/c-Src/EGFR/STAT5 signaling axis is a major regulator of tamoxifen-resistant breast cancer cell growth and survival.

Potent maturation of monocyte-derived dendritic cells after CD40L lentiviral gene delivery.

Dendritic cells (DCs) are being evaluated in immunization protocols to enhance immunity against infectious diseases and cancer. Interaction of T-helper cells expressing CD40 ligand (CD40L) with its cognate CD40 receptor on DCs leads to a mature DC phenotype, characterized by increased capacity of antigen presentation to cytotoxic T cells. The authors examined the ability of third-generation self-inactivating lentiviral vectors expressing CD40L to induce autonomous maturation of ex vivo expanded human monocyte-derived dendritic cells. Transduction with lentiviral vectors achieved a highly efficient gene transfer of CD40L to DCs, which correlated with phenotypic maturation as shown by the expression of immunologic relevant markers (CD83, CD80, MHCI) and secretion of IL-12, whereas DC phenotype was not affected by a control vector expressing only the green fluorescent protein marker. Addition of recombinant IFN-gamma to DCs at the time of CD40L transduction further enhanced IL-12 production, and when co-cultured with allogeneic and autologous CD8+ and CD4+ T cells, a potent activation was observed. Autologous responses against an HLA-A2-restricted influenza peptide (Flu-M1) and a tumor-associated antigenic peptide (gp100 210M) were significantly enhanced when CD40L transduced DCs were used as antigen-presenting cells for in vitro stimulation of CD8+ cytotoxic T lymphocytes. These results demonstrate that endogenous expression of CD40L by lentivirally transduced DCs induced their autonomous maturation to a phenotype comparable to that induced by optimal concentrations of soluble CD40L, providing a novel tool for genetic manipulation of DCs.

The use of lentiviral vectors in gene therapy of leukemia: combinatorial gene delivery of immunomodulators into leukemia cells by state-of-the-art vectors.

Our goal is to develop cell vaccines against leukemia cells, genetically modified to express molecules with potent immune-stimulatory capacities. Pre-clinical evaluation of this approach in murine models has demonstrated efficient anti-leukemic responses with the expression of immunomodulators, in particular GM-CSF and CD80, in irradiated cell vaccines. We have previously shown efficient insertion of GM-CSF and CD80 genes into primary human leukemia cells with the use of second and third generation self-inactivating (SIN) lentiviral vectors (Blood 96 (2000), 1317; Leukemia 16 (2002), 1645). The advantages of lentiviral vectors for development of autologous leukemia cell vaccines include: (1) efficient and consistent gene delivery; (2) high levels of transgene expression; (3) persistent expression of the transduced gene; (4) no viral proteins, as only the transduced gene is expressed; (5) no undesirable cytotoxic effects, and; (6) simplicity of use [leukemia cells are exposed to vector(s) only once]. In this work, we evaluated the insertion of the central polypurine tract and the central termination sequence into a SIN lentiviral vector encoding for GM-CSF and CD80, which significantly enhanced the transduction efficiency of primary leukemia cells and provided higher levels of GM-CSF and CD80 co-expression. We also demonstrate a methodology to deliver simultaneously a combination of immunomodulatory molecules (GM-CSF, CD80, IL-4, and CD40L) to activate different pathways of immune stimulation. Therefore, lentiviral vectors offer a simple, versatile, and reliable approach for engineering leukemic cells for use as cell vaccines.