Interplay between proteasome function and inflammatory responses in e-cig vapor condensate-challenged lung epithelial cells.

Exposure to cigarettes and other nicotine-based products results in persistent inflammation in the lung. In recent years, electronic cigarettes (e-cigs) have become extremely popular among adults and youth alike. E-cigarette vapor-induced oxidative stress promotes protein breakdown, DNA damage and cell death, culminating in a variety of respiratory diseases. The proteasome, a multi-catalytic protease, superintends protein degradation within the cell. When cells are stimulated with inflammatory cytokines such as IFN-γ and TNF-α, the constitutive catalytic proteasome subunits are replaced by the inducible subunits-low-molecular mass polypeptide (LMP)2 (ß1i), multi-catalytic endopeptidase complex-like (MECL)1 (ß2i), and LMP7 (ß5i), which are required for the production of certain MHC class I-restricted T-cell epitopes. In this study, we used human alveolar epithelial cells (A549) and exposed them to filtered air or (1%) tobacco-flavored (TF) electronic cigarette vapor condensate (ECVC) ± nicotine (6 mg/ml) (TF-ECVC ± N) for 24 h. We observed an increase in the levels of IFN-γ, TNF-α, and inducible proteasome subunits (LMP7/PSMB8, LMP2/PSMB9, MECL1/PSMB10), and a reduced expression of constitutive proteasome subunits (ß1/PSMB6 and ß2/PSMB7) in challenged A549 cells. Interestingly, knockdown of the inducible proteasome subunit LMP7 reversed ECVC-induced expression of NADPH oxidase and immunoproteasome subunits in A549 cells. In addition, pre-exposure to an LMP7 inhibitor (ONX-0914) abrogated the mRNA expression of several NOX subunits and rescued the excessive production/release of inflammatory cytokines/chemokines (IL-6, IL-8, CCL2, and CCL5) in ECVC-challenged cells. Our findings suggest an important role of LMP7 in regulating the expression of inflammatory mediators during ECVC exposure. Overall, our results provide evidence for proteasome-dependent ROS-mediated inflammation in ECVC-challenged cells.

Practical consensus recommendations on management of triple-negative metastatic breast cancer.

Patients with breast cancer along with metastatic estrogen and progesterone receptor (ER/PR)- and human epidermal growth factor receptor 2 (HER2)-negative tumors are referred to as having metastatic triple-negative breast cancer (mTNBC) disease. Resistance to current standard therapies such as anthracyclines or taxanes limits the available options for previously treated patients with metastatic TNBC to a small number of non-cross-resistant regimens, and there is currently no preferred standard chemotherapy. Clinical experience suggests that many women with triple-negative metastatic breast cancer (MBC) relapse quickly. Expert oncologist discussed about new chemotherapeutic strategies and agents used in treatment of mTNBC and the expert group used data from published literature, practical experience and opinion of a large group of academic oncologists to arrive at this practical consensus recommendations for the benefit of community oncologists.

Loss of claudin-3 expression induces IL6/gp130/Stat3 signaling to promote colon cancer malignancy by hyperactivating Wnt/ß-catenin signaling.

The hyperactivated Wnt/ß-catenin signaling acts as a switch to induce epithelial to mesenchymal transition and promote colorectal cancer. However, due to its essential role in gut homeostasis, therapeutic targeting of this pathway has proven challenging. Additionally, IL-6/Stat-3 signaling, activated by microbial translocation through the dysregulated mucosal barrier in colon adenomas, facilitates the adenoma to adenocarcinomas transition. However, inter-dependence between these signaling pathways and key mucosal barrier components in regulating colon tumorigenesis and cancer progression remains unclear. In current study, we have discovered, using a comprehensive investigative regimen, a novel and tissue-specific role of claudin-3, a tight junction integral protein, in inhibiting colon cancer progression by serving as the common rheostat of Stat-3 and Wnt-signaling activation. Loss of claudin-3 also predicted poor patient survival. These findings however contrasted an upregulated claudin-3 expression in other cancer types and implicated role of the epigenetic regulation. Claudin-3-/- mice revealed dedifferentiated and leaky colonic epithelium, and developed invasive adenocarcinoma when subjected to colon cancer. Wnt-signaling hyperactivation, albeit in GSK-3ß independent manner, differentiated colon cancer in claudin-3-/- mice versus WT-mice. Claudin-3 loss also upregulated the gp130/IL6/Stat3 signaling in colonic epithelium potentially assisted by infiltrating immune components. Genetic and pharmacological studies confirmed that claudin-3 loss induces Wnt/ß-catenin activation, which is further exacerbated by Stat-3-activation and help promote colon cancer. Overall, these novel findings identify claudin-3 as a therapeutic target for inhibiting overactivation of Wnt-signaling to prevent CRC malignancy.

Tuberculosis treatment outcomes among disadvantaged patients in India.

Setting: Urban slums and poor rural areas in India, 2012-2014. Objective: To describe the characteristics of tuberculosis (TB) patients enrolled in treatment through Operation ASHA, a non-governmental organisation serving disadvantaged populations in India, and to identify risk factors for unfavourable treatment outcomes. Design: This was a retrospective cohort study. Patient characteristics were assessed for their relationship with treatment outcomes using mixed effects logistic regression, adjusting for clustering by treatment centre and Indian state. Outcomes were considered favourable (cured/treatment completed) or unfavourable (treatment failure, loss to follow-up, death, switch to multidrug-resistant TB treatment, transfer out). Results: Of 8415 patients, 7148 (84.9%) had a favourable outcome. On multivariable analysis, unfavourable outcomes were more common among men (OR 1.31, 95%CI 1.15-1.51), older patients (OR 1.12, 95%CI 1.04-1.21) and previously treated patients (OR 2.05, 95%CI 1.79-2.36). Compared to pulmonary smear-negative patients, those with extra-pulmonary disease were less likely to have unfavourable outcomes (OR 0.72, 95%CI 0.60-0.87), while smear-positive pulmonary patients were more likely to have unfavourable outcomes (OR 1.38, 95%CI 1.15-1.66 for low [scanty/1+] and OR 1.71, 95%CI 1.44-2.04 for high [2+/3+] positive smears). Conclusion: The treatment success rate within Operation ASHA is comparable to that reported nationally for India. Men, older patients, retreatment cases and smear-positive pulmonary TB patients may need additional interventions to ensure a favourable outcome.

Contexte: Bidonvilles urbains et zones rurales pauvres, Inde, 2012­2014.Objectif: Décrire les caractéristiques des patients atteints de tuberculose (TB) enrôlés dans un traitement à travers l'Opération ASHA, une organisation non-gouvernementale au service des populations désavantagées en Inde, et identifier les facteurs de risque de résultat défavorable du traitement.Schéma: Etude rétrospective de cohorte. Les caractéristiques des patients ont été évaluées en fonction de leur relation avec les résultats du traitement grâce à une régression logistique à effets mixtes ajustée sur le regroupement par centre de traitement et par l'état d'Inde. Le résultat a été considéré comme favorable (guéri/traitement achevé) ou défavorable (échec du traitement, perte de vue, décès, évolution vers un traitement de TB multirésistante, transfert).Résultats: De 8415 patients, 7148 (84,9%) ont eu un résultat favorable. En analyse multi-variable, les résultats défavorables ont été plus fréquents parmi les hommes (OR 1,31 ; IC 95% 1,15­1,51), les patients plus âgés (OR 1,12 ; IC95% 1,04­1,21) et les patients déjà traités (OR 2,05 ; IC95% 1,79­2,36). Comparés aux patients atteints de TB pulmonaire à frottis négatif, les patients atteints de TB extra-pulmonaire ont été moins susceptibles d'avoir un résultat défavorable (OR 0,72 ; IC95% 0,60­0,87), tandis que les patients atteints de TB pulmonaire à frottis positif ont été plus susceptibles d'avoir un résultat défavorable (OR 1,38 ; IC95% 1,15­1,66 pour les frottis positifs faibles [rares/1+] et OR 1,71 ; IC95% 1,44­2,04 pour les frottis élevés [2+/3+]).Conclusion: Le taux de succès du traitement dans le cadre de l'Opération ASHA est comparable à celui rapporté au niveau national en Inde. Les hommes, les patients plus âgés, les cas en retraitement et les patients atteints de TB pulmonaire à frottis positif pourraient avoir besoin d'interventions supplémentaires afin d'assurer un résultat favorable.

Marco de referencia: Barriadas urbanas y zonas rurales pobres en la India del 2012 al 2014.Objetivo: Describir las características de los pacientes con tuberculosis (TB) inscritos en tratamiento en el marco de la Operación ASHA, que es una organización no gubernamental que atiende a las poblaciones desfavorecidas en la India, y determinar los factores de riesgo de obtener desenlaces terapéuticos desfavorables.Método: Un estudio retrospectivo de cohortes. Las características de los pacientes se evaluaron con respecto a los desenlaces terapéuticos, mediante un análisis de regresión logística de efectos mixtos y ajuste con relación a los conglomerados, según el centro de tratamiento y el estado de la India. Los desenlaces se consideraron favorables (curación o tratamiento completo) o desfavorables (fracaso terapéutico, pérdida durante el seguimiento, muerte, cambio de tratamiento por TB multirresistente o transferencia a otro centro).Resultados: De los 8415 pacientes, en 7148 el desenlace fue favorable (84,9%). El análisis multivariante reveló que los desenlaces desfavorables eran más frecuentes en los hombres (OR 1,31; IC95% 1,15­1,51), los ancianos (OR 1,12; IC95% 1,04­1,21) y en los pacientes con antecedente de tratamiento antituberculoso (OR 2,05; IC95% 1,79­2,36). Tomando como referencia a los pacientes con baciloscopia negativa, los desenlaces desfavorables fueron menos probables en los pacientes con TB extrapulmonar (OR 0,72; IC95% 0,60­0,87) y más probables en los pacientes con TB pulmonar y baciloscopia positiva (OR 1,38; IC95% 1,15­1,66 para las baciloscopias bajas, de escasos bacilos a 1+ y OR 1,71; IC95% 1,44­2,04 para las baciloscopia altas, de 2+ o 3+).Conclusión: La tasa de éxito del tratamiento antituberculoso en el marco de la Operación ASHA es equivalente a la notificada a escala nacional en la India. Los pacientes de sexo masculino, los ancianos, los casos en retratamiento y los que presentan una TB pulmonar con baciloscopia positiva pueden necesitar intervenciones complementarias a fin de fomentar los desenlaces favorables.

Gut permeability and mucosal inflammation: bad, good or context dependent.

Inflammatory bowel disease (IBD) is a multifactorial disease. A breach in the mucosal barrier, otherwise known as "leaky gut," is alleged to promote mucosal inflammation by intensifying immune activation. However, interaction between the luminal antigen and mucosal immune system is necessary to maintain mucosal homeostasis. Furthermore, manipulations leading to deregulated gut permeability have resulted in susceptibility in mice to colitis as well as to creating adaptive immunity. These findings implicate a complex but dynamic association between mucosal permeability and immune homeostasis; however, they also emphasize that compromised gut permeability alone may not be sufficient to induce colitis. Emerging evidence further supports the role(s) of proteins associated with the mucosal barrier in epithelial injury and repair: manipulations of associated proteins also modified epithelial differentiation, proliferation, and apoptosis. Taken together, the role of gut permeability and proteins associated in regulating mucosal inflammatory diseases appears to be more complex than previously thought. Herein, we review outcomes from recent mouse models where gut permeability was altered by direct and indirect effects of manipulating mucosal barrier-associated proteins, to highlight the significance of mucosal permeability and the non-barrier-related roles of these proteins in regulating chronic mucosal inflammatory conditions.

Demographics of tuberculosis of spine and factors affecting neurological improvement in patients suffering from tuberculosis of spine: a retrospective analysis of 312 cases.

OBJECTIVES: The purpose of the study was to study demographics of tuberculosis of spine and analyze factors that might affect neurological improvement in such patients. METHODS: Of the 638 suspected cases of spinal tuberculosis, 312 cases with confirmed diagnosis with at least 1-year follow-up were selected for retrospective analysis. Two hundred cases who presented with neurological deficit were further divided into three groups-completely improved, partially improved and no improvement according to American Spinal Injury Association impairment scale (AIS) grading. All continuous variables and categorical variables were compared across groups. RESULTS: A total of 209 (66.99%) patients had typical clinical presentation. A total of 264 (84.62%) had typical magnetic resonance imaging (MRI) presentation. Among 356 involved vertebrae, thoracic levels (T1-10) were most commonly affected in 163 (45.78%) followed by thoracolumbar (T11-L2) vertebrae in 98 (27.52%). In 250 patients (80.12%), disease was restricted to one or two adjoining vertebrae. At presentation, 112 (35.89%) patients were neurologically intact, whereas 97 (31%) were AIS D, 65 (20.83%) were AIS C, 8 (2.5%) were AIS B and 30 (9.61%) were AIS A. On statistical analysis, although three groups of patients with complete improvement, partial improvement and no improvement were similar in age, sex, radiological presentation, and co-morbidities and the presence of pulmonary tuberculosis, they were significantly different with regard to the levels of vertebral involvement, AIS grade at presentation, bladder and bowel involvement and its duration. CONCLUSIONS: In management of patients suffering from tuberculosis of spine, levels of vertebral involvement, AIS grade at presentation, bladder and bowel involvement and its duration significantly affect the final neurological improvement.

RAC1 GTPase promotes the survival of breast cancer cells in response to hyper-fractionated radiation treatment.

Radiation therapy is a staple approach for cancer treatment, whereas radioresistance of cancer cells remains a substantial clinical problem. In response to ionizing radiation (IR) induced DNA damage, cancer cells can sustain/activate pro-survival signaling pathways, leading to apoptotic resistance and induction of cell cycle checkpoint/DNA repair. Previous studies show that Rac1 GTPase is overexpressed/hyperactivated in breast cancer cells and is associated with poor prognosis. Studies from our laboratory reveal that Rac1 activity is necessary for G2/M checkpoint activation and cell survival in response to IR exposure of breast and pancreatic cancer cells. In this study, we investigated the effect of Rac1 on the survival of breast cancer cells treated with hyper-fractionated radiation (HFR), which is used clinically for cancer treatment. Results in this report indicate that Rac1 protein expression is increased in the breast cancer cells that survived HFR compared with parental cells. Furthermore, this increase of Rac1 is associated with enhanced activities of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and nuclear factor-κB (NF-κB) signaling pathways and increased levels of anti-apoptotic protein Bcl-xL and Mcl-1, which are downstream targets of ERK1/2 and NF-κB signaling pathways. Using Rac1-specific inhibitor and dominant-negative mutant N17Rac1, here we demonstrate that Rac1 inhibition decreases the phosphorylation of ERK1/2 and inhibitory κBα (IκBα), as well as the levels of Bcl-xL and Mcl-1 protein in the HFR-selected breast cancer cells. Moreover, inhibition of Rac1 using either small molecule inhibitor or dominant-negative N17Rac1 abrogates clonogenic survival of HFR-selected breast cancer cells and decreases the level of intact poly(ADP-ribose) polymerase, which is indicative of apoptosis induction. Collectively, results in this report suggest that Rac1 signaling is essential for the survival of breast cancer cells subjected to HFR and implicate Rac1 in radioresistance of breast cancer cells. These studies also provide the basis to explore Rac1 as a therapeutic target for radioresistant breast cancer cells.

Hypoxia-induced oxidative stress promotes MUC4 degradation via autophagy to enhance pancreatic cancer cells survival.

Pancreatic cancer (PC) and associated pre-neoplastic lesions have been reported to be hypoxic, primarily due to hypovascular nature of PC. Though the presence of hypoxia under cancerous condition has been associated with the overexpression of oncogenic proteins (MUC1), multiple emerging reports have also indicated the growth inhibitory effects of hypoxia. In spite of being recognized as the top-most differentially expressed and established oncogenic protein in PC, MUC4 regulation in terms of micro-environmental stress has not been determined. Herein, for the first time, we are reporting that MUC4 protein stability is drastically affected in PC, under hypoxic condition in a hypoxia inducible factor 1α (HIF-1α)-independent manner. Mechanistically, we have demonstrated that hypoxia-mediated induction of reactive oxygen species (ROS) promotes autophagy by inhibiting pAkt/mTORC1 pathway, one of the central regulators of autophagy. Immunohistofluorescence analyses revealed significant negative correlation (P-value=0.017) between 8-hydroxy guanosine (8-OHG) and MUC4 in primary pancreatic tumors (n=25). Moreover, we found pronounced colocalization between MUC4 and LAMP1/LC3 (microtubule-associated protein 1A/1B-light chain 3) in PC tissues and also observed their negative relationship in their expression pattern, suggesting that areas with high autophagy rate had less MUC4 expression. We also found that hypoxia and ROS have negative impact on overall cell growth and viability, which was partially, though significantly (P<0.05), rescued in the presence of MUC4. Altogether, hypoxia-mediated oxidative stress induces autophagy in PC, leading to the MUC4 degradation to enhance survival, possibly by offering required metabolites to stressed cells.

MUC5AC interactions with integrin ß4 enhances the migration of lung cancer cells through FAK signaling.

MUC5AC is a secretory mucin aberrantly expressed in various cancers. In lung cancer, MUC5AC is overexpressed in both primary and metastatic lesions; however, its functional role is not well understood. The present study was aimed at evaluating mechanistic role of MUC5AC on metastasis of lung cancer cells. Clinically, the overexpression of MUC5AC was observed in lung cancer patient tissues and was associated with poor survival. In addition, the overexpression of Muc5ac was also observed in genetically engineered mouse lung adenocarcinoma tissues (Kras(G12D); Trp53(R172H/+); AdCre) in comparison with normal lung tissues. Our functional studies showed that MUC5AC knockdown resulted in significantly decreased migration in two lung cancer cell lines (A549 and H1437) as compared with scramble cells. Expression of integrins (α5, ß1, ß3, ß4 and ß5) was decreased in MUC5AC knockdown cells. As both integrins and MUC5AC have a von Willebrand factor domain, we assessed for possible interaction of MUC5AC and integrins in lung cancer cells. MUC5AC strongly interacted only with integrin ß4. The co-localization of MUC5AC and integrin ß4 was observed both in A549 lung cancer cells as well as genetically engineered mouse adenocarcinoma tissues. Activated integrins recruit focal adhesion kinase (FAK) that mediates metastatic downstream signaling pathways. Phosphorylation of FAK (Y397) was decreased in MUC5AC knockdown cells. MUC5AC/integrin ß4/FAK-mediated lung cancer cell migration was confirmed through experiments utilizing a phosphorylation (Y397)-specific FAK inhibitor. In conclusion, overexpression of MUC5AC is a poor prognostic marker in lung cancer. MUC5AC interacts with integrin ß4 that mediates phosphorylation of FAK at Y397 leading to lung cancer cell migration.

IFN-γ induction by neutrophil-derived IL-17A homodimer augments pulmonary antibacterial defense.

The role of interleukin-17A (IL-17A) in host defense against Legionella pneumophila remains elusive. To address this issue, we used Il17a(-/-), Il17f(-/-), and Il17a/Il17f(-/-) mice on a C57Bl/6 (non-permissive) background and IL-17 neutralizing Abs in mice on an A/J (permissive) background. Higher bacterial (L. pneumophila) counts in the lung and blood along with reduced neutrophil recruitment were detected in Il17a(-/-), but not Il17f(-/-), mice. We found that neutrophils produce IL-17A homodimer (IL-17A) during L. pneumophila infection, and hematopoietic cell-derived IL-17A is known to be important for bacterial clearance. Thus, intratracheal administration of wild-type neutrophils or recombinant IL-17A restored bacterial clearance and neutrophil recruitment in Il17a(-/-) mice. Furthermore, neutrophil-depleted Rag2(-/-) and Rag2/Il-2rγ(-/-) mice exhibited increased bacterial burden, reduced neutrophil influx and IL-17A production in the lung. Recombinant IFN-γ administration in Il17a(-/-) mice augmented bacterial elimination, whereas IL-17A administration in Ifnγ(-/-) mice did not augment bacterial clearance. IFN-γ is produced by T cells, but not neutrophils or macrophages, suggesting that neutrophil-derived IL-17A induces IFN-γ in a paracrine fashion. Human pneumonic lungs and human neutrophils challenged with L. pneumophila exhibited increased numbers of IL-17A producing cells. These findings display a novel function of neutrophil-derived IL-17A in antibacterial defense via the induction of IFN-γ in a paracrine manner.

NLRP12 modulates host defense through IL-17A-CXCL1 axis.

We used an extracellular pathogen Klebsiella pneumoniae to determine the role of NLRP12 (NOD-like receptor (NLR) family pyrin domain containing 12) as this bacterium is associated with devastating pulmonary infections. We found that human myeloid cells (neutrophils and macrophages) and non-myeloid cells (epithelial cells) show upregulation of NLRP12 in human pneumonic lungs. NLRP12-silenced human macrophages and murine Nlrp12(-/-) macrophages displayed reduced activation of nuclear factor-κB and mitogen-activated protein kinase, as well as expression of histone deacetylases following K. pneumoniae infection. NLRP12 is important for the production of interleukin-1ß (IL-1ß) in human and murine macrophages following K. pneumoniae infection. Furthermore, host survival, bacterial clearance, and neutrophil recruitment are dependent on NLRP12 following K. pneumoniae infection. Using bone marrow chimeras, we showed that hematopoietic cell-driven NLRP12 signaling predominantly contributes to host defense against K. pneumoniae. Intratracheal administration of either IL-17A+ CD4 T cells or chemokine (C-X-C motif) ligand 1 (CXCL1+) macrophages rescues host survival, bacterial clearance, and neutrophil recruitment in Nlrp12(-/-) mice following K. pneumoniae infection. These novel findings reveal the critical role of NLRP12-IL-17A-CXCL1 axis in host defense by modulating neutrophil recruitment against this extracellular pathogen.

Mice deficient in Muc4 are resistant to experimental colitis and colitis-associated colorectal cancer.

MUC4, a large transmembrane mucin normally expressed in the small and large intestine, is differentially expressed during inflammatory and malignant conditions of the colon. However, the expression pattern and the role of MUC4 in colitis and colorectal cancer (CRC) are inconclusive. Therefore, the aim of this study was to understand the role of Muc4 during inflammatory and malignant conditions of the colon. Here, we generated Muc4(-/-) mice and addressed its role in colitis and colitis-associated CRC using dextran sodium sulfate (DSS) and azoxymethane (AOM)-DSS experimental models, respectively. Muc4(-/-) mice were viable, fertile with no apparent defects. Muc4(-/-) mice displayed increased resistance to DSS-induced colitis compared with wild-type (WT) littermates that was evaluated by survival rate, body weight loss, diarrhea and fecal blood score, and histological score. Reduced infiltration of inflammatory cells, that is, CD3(+) lymphocytes and F4/80(+) macrophages was observed in the inflamed mucosa along with reduction in the mRNA levels of inflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor (TNF)-α and anti-microbial genes Lysozyme M and SLPI in the colon of Muc4(-/-) mice compared with WT littermates. Compensatory upregulation of Muc2 and Muc3 mucins under basal and DSS treatment conditions partly explains the resistance observed in Muc4(-/-) mice. Accordingly, Muc4(-/-) mice exhibited significantly reduced tumor burden compared with WT mice assessed in a colitis-induced tumor model using AOM/DSS. An increased percentage of Ki67(+) nuclei was observed in the tumors from WT compared with Muc4(-/-) mice suggesting Muc4 to be critical in intestinal cell proliferation during tumorigenesis. Taken together, we conclusively demonstrate for the first time the role of Muc4 in driving intestinal inflammation and inflammation-associated tumorigenesis using a novel Muc4(-/-) mouse model.

Development and evaluation of a standardized method and atlas for contouring primary and permanent dentition.

OBJECTIVES: Radiation toxicity of the dentition may present significant treatment-related morbidity in the paediatric head and neck cancer population. However, clear dose-effect relationships remain undetermined and must be predicated upon accurate structure delineation and dosimetry at the individual tooth level. Radiation oncologists generally have limited familiarity or experience with relevant dental anatomy. METHODS: We therefore developed a detailed CT atlas of permanent and primary dentition. After studying this atlas, five radiation oncology clinicians delineated all teeth for each of eight different cases (selected for breadth of dental maturity and anatomical variability). They were asked to record confidence in their contours on a per-tooth basis as well as the duration of time required per case. Contour accuracy and interclinician variability were assessed by Hausdorff distance and Dice similarity coefficient. All analyses were performed using R v. 3.1.1 and the RadOnc v. 1.0.9 package. RESULTS: Participating clinicians delineated teeth with varying degrees of completeness and accuracy, stratified primarily by the age of the subject. On a per-tooth basis, delineation of permanent dentition was feasible for incisors, canines, premolars and first molars among all subjects, even at the youngest ages. However, delineation of second and third molars was less consistent, commensurate with approximate timing of tooth development. Within each tooth contour, uncertainty was the greatest at the level of the dental roots. CONCLUSIONS: Delineation of individual teeth is feasible and serves as a necessary precursor for dental dose assessment and avoidance. Among the paediatric radiation oncology community in particular, this atlas may serve as a useful tool and reference.

NCOA3-mediated upregulation of mucin expression via transcriptional and post-translational changes during the development of pancreatic cancer.

Pancreatic cancer (PC) is characterized by aberrant overexpression of mucins that contribute to its pathogenesis. Although the inflammatory cytokines contribute to mucin overexpression, the mucin profile of PC is markedly distinct from that of normal or inflamed pancreas. We postulated that de novo expression of various mucins in PC involves chromatin modifications. Analysis of chromatin modifying enzymes by PCR array identified differential expression of NCOA3 in MUC4-expressing PC cell lines. Immunohistochemistry analysis in tumor tissues from patients and spontaneous mouse models, and microarray analysis following the knockdown of NCOA3 were performed to elucidate its role in mucin regulation and overall impact on PC. Silencing of NCOA3 in PC cell lines resulted in significant downregulation of two most differentially expressed mucins in PC, MUC4 and MUC1 (P<0.01). Immunohistochemistry analysis in PC tissues and metastatic lesions established an association between NCOA3 and mucin (MUC1 and MUC4) expression. Spontaneous mouse model of PC (K-ras(G12D); Pdx-1cre) showed early expression of Ncoa3 during pre-neoplastic lesions. Mechanistically, NCOA3 knockdown abrogated retinoic acid-mediated MUC4 upregulation by restricting MUC4 promoter accessibility as demonstrated by micrococcus nuclease digestion (P<0.05) and chromatin immuno-precipitation analysis. NCOA3 also created pro-inflammatory conditions by upregulating chemokines like CXCL1, 2, 5 and CCL20 (P<0.001). AKT, ubiquitin C, ERK1/2 and NF-κB occupied dominant nodes in the networks significantly modulated after NCOA3 silencing. In addition, NCOA3 stabilized mucins post translationally through fucosylation by FUT8, as the knockdown of FUT8 resulted in the downregulation of MUC4 and MUC1 at protein levels.

MUC4 regulates cellular senescence in head and neck squamous cell carcinoma through p16/Rb pathway.

The limited effectiveness of therapy for patients with advanced stage head and neck squamous cell carcinoma (HNSCC) or recurrent disease is a reflection of an incomplete understanding of the molecular basis of HNSCC pathogenesis. MUC4, a high molecular weight glycoprotein, is differentially overexpressed in many human cancers and implicated in cancer progression and resistance to several chemotherapies. However, its clinical relevance and the molecular mechanisms through which it mediates HNSCC progression are not well understood. This study revealed a significant upregulation of MUC4 in 78% (68/87) of HNSCC tissues compared with 10% positivity (1/10) in benign samples (P=0.006, odds ratio (95% confidence interval)=10.74 (2.0-57.56). MUC4 knockdown (KD) in SCC1 and SCC10B HNSCC cell lines resulted in significant inhibition of growth in vitro and in vivo, increased senescence as indicated by an increase in the number of flat, enlarged and senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells. Decreased cellular proliferation was associated with G0/G1 cell cycle arrest and decrease expression of cell cycle regulatory proteins like cyclin E, cyclin D1 and decrease in BrdU incorporation. Mechanistic studies revealed upregulation of p16, pRb dephosphorylation and its interaction with histone deacetylase 1/2. This resulted in decreased histone acetylation (H3K9) at cyclin E promoter leading to its downregulation. Orthotopic implantation of MUC4 KD SCC1 cells into the floor of the mouth in nude mice resulted in the formation of significantly smaller tumors (170±18.30 mg) compared to those (375±17.29 mg) formed by control cells (P=0.00007). In conclusion, our findings showed that MUC4 overexpression has a critical role by regulating proliferation and cellular senescence of HNSCC cells. Downregulation of MUC4 may be a promising therapeutic approach for treating HNSCC patients.

Smoking accelerates pancreatic cancer progression by promoting differentiation of MDSCs and inducing HB-EGF expression in macrophages.

Smoking is an established risk factor for pancreatic cancer (PC), but late diagnosis limits the evaluation of its mechanistic role in the progression of PC. We used a well-established genetically engineered mouse model (LSL-K-ras(G12D)) of PC to elucidate the role of smoking during initiation and development of pancreatic intraepithelial neoplasia (PanIN). The 10-week-old floxed mice (K-ras(G12D); Pdx-1cre) and their control unfloxed (LSL-K-ras(G12D)) littermates were exposed to cigarette smoke (total suspended particles: 150 mg/m(3)) for 20 weeks. Smoke exposure significantly accelerated the development of PanIN lesions in the floxed mice, which correlated with tenfold increase in the expression of cytokeratin19. The systemic accumulation of myeloid-derived suppressor cells (MDSCs) decreased significantly in floxed mice compared with unfloxed controls (P<0.01) after the smoke exposure with the concurrent increase in the macrophage (P<0.05) and dendritic cell (DCs) (P<0.01) population. Further, smoking-induced inflammation (IFN-γ, CXCL2; P<0.05) was accompanied by enhanced activation of pancreatic stellate cells and elevated levels of serum retinoic acid-binding protein 4, indicating increased bioavailability of retinoic acid which contributes to differentiation of MDSCs to tumor-associated macrophages (TAMs) and DCs. TAMs predominantly contribute to the increased expression of heparin-binding epidermal growth factor-like growth factor (EGFR ligand) in pre-neoplastic lesions in smoke-exposed floxed mice that facilitate acinar-to-ductal metaplasia (ADM). Further, smoke exposure also resulted in partial suppression of the immune system early during PC progression. Overall, the present study provides a novel mechanism of smoking-induced increase in ADM in the presence of constitutively active K-ras mutation.

A novel function of HER2/Neu in the activation of G2/M checkpoint in response to γ-irradiation.

In response to γ-irradiation (IR)-induced DNA damage, activation of cell cycle checkpoints results in cell cycle arrest, allowing time for DNA repair before cell cycle re-entry. Human cells contain G1 and G2 cell cycle checkpoints. While G1 checkpoint is defective in most cancer cells, commonly due to mutations and/or alterations in the key regulators of G1 checkpoint (for example, p53, cyclin D), G2 checkpoint is rarely impaired in cancer cells, which is important for cancer cell survival. G2 checkpoint activation involves activation of ataxia telangiectasia-mutated (ATM)/ATM- and rad3-related (ATR) signalings, which leads to the inhibition of Cdc2 kinase and subsequent G2/M cell cycle arrest. Previous studies from our laboratory show that G2 checkpoint activation following IR exposure of MCF-7 breast cancer cells is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. As HER receptor tyrosine kinases (RTKs), which have important roles in cell proliferation and survival, have been shown to activate ERK1/2 signaling in response to various stimuli, we investigated the role of HER RTKs in IR-induced G2/M checkpoint response in breast cancer cells. Results of the present studies indicate that IR exposure resulted in a striking increase in the phosphorylation of HER1, HER2, HER3 and HER4 in MCF-7 cells, indicative of activation of these proteins. Furthermore, specific inhibition of HER2 using an inhibitor, short hairpin RNA and dominant-negative mutant HER2 abolished IR-induced activation of ATM/ATR signaling, phosphorylation of Cdc2-Y15 and subsequent induction of G2/M arrest. Moreover, the inhibition of HER2 also abrogated IR-induced ERK1/2 phosphorylation. In contrast, inhibition of HER1 using specific inhibitors or decreasing expression of HER3 or HER4 using short hairpin RNAs did not block the induction of G2/M arrest following IR. These results suggest an important role of HER2 in the activation of G2/M checkpoint response following IR.

Unbiased analysis of pancreatic cancer radiation resistance reveals cholesterol biosynthesis as a novel target for radiosensitisation.

BACKGROUND: Despite its promise as a highly useful therapy for pancreatic cancer (PC), the addition of external beam radiation therapy to PC treatment has shown varying success in clinical trials. Understanding PC radioresistance and discovery of methods to sensitise PC to radiation will increase patient survival and improve quality of life. In this study, we identified PC radioresistance-associated pathways using global, unbiased techniques. METHODS: Radioresistant cells were generated by sequential irradiation and recovery, and global genome cDNA microarray analysis was performed to identify differentially expressed genes in radiosensitive and radioresistant cells. Ingenuity pathway analysis was performed to discover cellular pathways and functions associated with differential radioresponse and identify potential small-molecule inhibitors for radiosensitisation. The expression of FDPS, one of the most differentially expressed genes, was determined in human PC tissues by IHC and the impact of its pharmacological inhibition with zoledronic acid (ZOL, Zometa) on radiosensitivity was determined by colony-forming assays. The radiosensitising effect of Zol in vivo was determined using allograft transplantation mouse model. RESULTS: Microarray analysis indicated that 11 genes (FDPS, ACAT2, AG2, CLDN7, DHCR7, ELFN2, FASN, SC4MOL, SIX6, SLC12A2, and SQLE) were consistently associated with radioresistance in the cell lines, a majority of which are involved in cholesterol biosynthesis. We demonstrated that knockdown of farnesyl diphosphate synthase (FDPS), a branchpoint enzyme of the cholesterol synthesis pathway, radiosensitised PC cells. FDPS was significantly overexpressed in human PC tumour tissues compared with healthy pancreas samples. Also, pharmacologic inhibition of FDPS by ZOL radiosensitised PC cell lines, with a radiation enhancement ratio between 1.26 and 1.5. Further, ZOL treatment resulted in radiosensitisation of PC tumours in an allograft mouse model. CONCLUSIONS: Unbiased pathway analysis of radioresistance allowed for the discovery of novel pathways associated with resistance to ionising radiation in PC. Specifically, our analysis indicates the importance of the cholesterol synthesis pathway in PC radioresistance. Further, a novel radiosensitiser, ZOL, showed promising results and warrants further study into the universality of these findings in PC, as well as the true potential of this drug as a clinical radiosensitiser.

MELD exceptions for portopulmonary hypertension: current policy and future implementation.

Since 2006, waitlist candidates with portopulmonary hypertension (POPH) have been eligible for standardized Model for End-Stage Liver Disease (MELD) exception points. However, there are no data evaluating the current POPH exception policy and its implementation. We used Organ Procurement and Transplantation Network (OPTN) data to compare outcomes of patients with approved POPH MELD exceptions from 2006 to 2012 to all nonexception waitlist candidates during this period. Since 2006, 155 waitlist candidates had approved POPH MELD exceptions, with only 73 (47.1%) meeting the formal OPTN exception criteria. Furthermore, over one-third of those with approved POPH exceptions either did not fulfill hemodynamic criteria consistent with POPH or had missing data, with 80% of such patients receiving a transplant based on receiving exception points. In multivariable multistate survival models, waitlist candidates with POPH MELD exceptions had an increased risk of death compared to nonexception waitlist candidates, regardless of whether they did (hazard ratio [HR]: 2.46, 95% confidence interval [CI]: 1.73-3.52; n = 100) or did not (HR: 1.60, 95% CI: 1.04-2.47; n = 55) have hemodynamic criteria consistent with POPH. These data highlight the need for OPTN/UNOS to reconsider not only the policy for POPH MELD exceptions, but also the process by which such points are awarded.