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Chronic post-surgical pain (CPSP) is a highly prevalent complication following thoracic surgery. This is a prospective cohort study that aims to describe the pain trajectories of patients undergoing thoracic surgery beginning preoperatively and up to 1 year after surgery Two hundred and seventy nine patients undergoing elective thoracic surgery were enrolled. Participants filled out a preoperative questionnaire containing questions about their sociodemographic information, comorbidities as well as several psychological and pain-related statuses. They were then followed-up during their immediate postoperative period and at the three, six and 12 month time-points to track their postoperative pain, complications and pain-related outcomes. Growth mixture modeling was used to construct pain trajectories. The first trajectory is characterized by 185 patients (78.1%) with mild pain intensity across the 12 month period. The second is characterized by 32 patients (7.5%) with moderate pain intensity immediately after surgery which decreases markedly by 3 months and remains low at the 12 month follow-up. The final trajectory is characterized by 20 patients (8.4%) with moderate pain intensity immediately after surgery which persists at 12 months. Patients with moderate to severe postoperative pain intensity were much more likely to develop CPSP compared to patients with mild pain intensity. Initial pain intensity levels immediately following surgery as well as levels of pain catastrophizing at baseline were predicting pain trajectory membership. None of the surgical or anesthetic-related variables were significantly associated with pain trajectory membership. Patients who undergo thoracic surgery can have postoperative pain that follows one of the three different types of trajectories. Higher levels of immediate postoperative pain and preoperative pain catastrophizing were associated with moderately severe CPSP.

INTRODUCTION: Successfully targeting aberrant Wnt signaling in cancer, and even more so in chronic diseases, requires a fine balancing act, wherein the ‘dark side’ of Wnt signaling in disease can be abrogated without interfering with the crucial roles of Wnt signaling in normal tissue homeostasis and repair. Our identification of small molecule CBP/β-Catenin antagonists revealed (a) the distinctive roles that the Kat3 coactivators CBP and p300 play in Wnt signaling and orchestrating critical cellular decisions, and (b) their therapeutic potential. AREAS COVERED: This perspective discusses the challenges of targeting the Wnt signaling cascade, the safety, efficacy, and therapeutic potential of specific CBP/β-catenin antagonists and a rationale for the pleiotropic effects of CBP/β-catenin antagonists beyond Wnt signaling. EXPERT OPINION: CBP/β-catenin antagonists can correct lineage infidelity, enhance wound healing, both normal and aberrant (e.g. fibrosis) and force the differentiation and lineage commitment of stem cells and cancer stem cells by regulating enhancer and super-enhancer coactivator occupancy. Small molecule CBP/β-catenin antagonists rebalance the equilibrium between CBP/β-catenin versus p300/β-catenin dependent transcription and may be able to treat or prevent many diseases of aging, via maintenance of our somatic stem cell pool, and regulating mitochondrial function and metabolism involved in differentiation and immune cell function.

WNT/β-catenin signaling plays fundamental roles in numerous developmental processes and in adult tissue homeostasis and repair after injury, by controlling cellular self-renewal, activation, division, differentiation, movement, genetic stability, and apoptosis. As such, it comes as no surprise that dysregulation of WNT/β-catenin signaling is associated with various diseases, including cancer, fibrosis, neurodegeneration, etc. Although multiple agents that specifically target the WNT/β-catenin signaling pathway have been studied preclinically and a number have entered clinical trials, none has been approved by the FDA to date. In this chapter, we provide our insights as to the reason(s) it has been so difficult to safely pharmacologically target the WNT/β-catenin signaling pathway and discuss the significant efforts undertaken towards this goal.

Highlights • Patient tumor subclones that survive chemotherapy acquire primitive cell traits. • HDAC inhibitors can reverse chemo-acquired stemness states and abolish self-renewal abilities. • Belinostat promotes stem cell differentiation and inhibits HDAC and MYC pathways. • A ‘one-two punch’, chemotherapy-HDAC inhibitor combination strategy reverses chemo-induced resistant phenotypes., Cancer cell phenotypes evolve during a tumor's treatment. In some cases, tumor cells acquire cancer stem cell-like (CSL) traits such as resistance to chemotherapy and diminished differentiation; therefore, targeting these cells may be therapeutically beneficial. In this study we show that in progressive estrogen receptor positive (ER+) metastatic breast cancer tumors, resistant subclones that emerge following chemotherapy have increased CSL abundance. Further, in vitro organoid growth of ER+ patient cancer cells also shows that chemotherapy treatment leads to increased abundance of ALDH+/CD44+ CSL cells. Chemotherapy induced CSL abundance is blocked by treatment with a pan-HDAC inhibitor, belinostat. Belinostat treatment diminished both mammosphere formation and size following chemotherapy, indicating a decrease in progenitor CSL traits. HDAC inhibitors specific to class IIa (HDAC4, HDAC5) and IIb (HDAC6) were shown to primarily reverse the chemo-resistant CSL state. Single-cell RNA sequencing analysis with patient samples showed that HDAC targets and MYC signaling were promoted by chemotherapy and inhibited upon HDAC inhibitor treatment. In summary, HDAC inhibition can block chemotherapy-induced drug resistant phenotypes with ‘one-two punch’ strategy in refractory breast cancer cells., Graphical abstract Image, graphical abstract

Background The prognostic implication of wild-type APC (APC-WT) in microsatellite stable (MSS) metastatic colorectal cancer (mCRC) is not well defined. Materials and Methods APC prognostic value was evaluated retrospectively in two independent cohorts of patient with MSS mCRC with a confirmatory analysis from a public data set from Memorial Sloan Kettering Cancer Center (MSKCC). Results In comparison with the APC-mutant (APC-MT) population (n = 255), APC-WT patients (n = 86) tended to be younger (59% of age 50), right-sided (41.7% vs. 27%), BRAFV600E mutated (23.3% vs. 0.8%), and KRAS wild type (65.1% vs. 49.8%). Alternative WNT pathway alterations, RNF43 and CTNNB1, were over-represented in the APC-WT versus APC-MT population (7% vs. 0.4% and 4.7% vs. 0.4%, respectively). APC-WT patients had a worse overall survival (OS) than APC-MT patients (22.6 vs. 45.6 months, p < .0001). Using a multivariate model correcting for primary tumor location, RAS and BRAF status, APC-WT was predictive of poor survival (APC-MT vs. APC-WT, hazard ratio [HR], 0.62; 95% confidence interval [CI], 0.44–0.86, p = .0037). The prognostic implication of APC-WT on OS was confirmed further in a similar multivariate model of 934 stage IV patients from MSKCC public database (APC-MT vs. APC-WT, HR, 0.63, 95% CI, 0.49–0.81, p < .0001). Conclusion APC-WT is associated with poor OS in MSS mCRC regardless of RAS and BRAF status. Compared with APC-MT mCRC tumors, APC-WT tumors were associated with other Wnt activating alterations, including RNF43 and CTNBB1. Our data suggest alternative therapy needs to be investigated in APC-WT patients. Implications for Practice Patients with microsatellite stable metastatic colorectal cancer with wild-type APC had a worse overall survival than patients with mutated APC regardless of RAS/RAF status. APC status should be considered as a stratification factor in prospective trials, and novel therapeutic strategies need to be developed for this subgroup of patients.

Objective Mobile health platforms have become an important component of pain self-management programs and hundreds of mobile applications are commercially available for patients to monitor pain. However, few of these applications have been developed in collaboration with healthcare professionals or have been critically evaluated. Manage My Pain is a user-driven mobile health platform developed by ManagingLife in collaboration with clinician researchers. Manage My Pain allows patients to keep a “pain record” and supports communication of this information with clinicians. The current report describes a user engagement study of Manage My Pain among patients at the Transitional Pain Service (TPS) at Toronto General Hospital, a multidisciplinary clinic for patients at high risk of developing postsurgical pain. Methods Patients at the TPS were encouraged to register on Manage My Pain as one component of a larger, non-randomized prospective study of treatment predictors and treatment enhancement. Uptake of the application and rates of registration, use, and retention were tracked for 90 days. Results Of the 196 patients who consented to the larger study, 132 (67%) also provided consent to the Manage My Pain component, indicating that they found this to be an acceptable treatment adjunct, and 119 (61%) completed registration. Of those who used the app, 67.9% and 43.2% continued to use Manage My Pain beyond 30 and 90 days, respectively. On average, users engaged with the app for 93.14 days (SD = 151.9 days) logged an average of 47.39 total records (SD = 136.1). Conclusions Manage My Pain was found acceptable by a majority of patients at an academic pain management program. Rates of user registration and retention were favorable compared to those reported by other applications. Further research is needed to develop strategies to retain users and maximize patient benefit.

The Wnt signaling pathway is one of the major signaling pathways used by cancer stem cells (CSC). Ecotropic Viral Integration Site 1 (EVI1) has recently been shown to regulate oncogenic development of tumor cells by interacting with multiple signaling pathways, including the Wnt signaling. In the present study, we found that the Wnt modulator ICG-001 could inhibit the expression of EVI1 in nasopharyngeal carcinoma (NPC) cells. Results from loss-of-function and gain-of-function studies revealed that EVI1 expression positively regulated both NPC cell migration and growth of CSC-enriched tumor spheres. Subsequent studies indicated ICG-001 inhibited EVI1 expression via upregulated expression of miR-96. Results from EVI1 3&prime, UTR luciferase reporter assay confirmed that EVI1 is a direct target of miR-96. Further mechanistic studies revealed that ICG-001, overexpression of miR-96, or knockdown of EVI1 expression could restore the expression of miR-449a. The suppressive effect of miR-449a on the cell migration and tumor sphere formation was confirmed in NPC cells. Taken together, the miR-96/EVI1/miR-449a axis is a novel pathway involved in ICG-001-mediated inhibition of NPC cell migration and growth of the tumor spheres.

Wnt signaling is a major driver of stemness and chemoresistance in ovarian cancer, yet the genetic drivers that stimulate its expression remain largely unknown. Unlike other cancers, mutations in the Wnt pathway are not reported in high-grade serous ovarian cancer (HGSOC). Hence, a key challenge that must be addressed to develop effective targeted therapies is to identify nonmutational drivers of Wnt activation. Using an miRNA sensor-based approach, we have identified miR-181a as a novel driver of Wnt/β-catenin signaling. miR-181ahigh primary HGSOC cells exhibited increased Wnt/β-catenin signaling, which was associated with increased stem-cell frequency and platinum resistance. Consistent with these findings, inhibition of β-catenin decreased stem-like properties in miR-181ahigh cell populations and downregulated miR-181a. The Wnt inhibitor SFRP4 was identified as a novel target of miR-181a. Overall, our results demonstrate that miR-181a is a nonmutational activator of Wnt signaling that drives stemness and chemoresistance in HGSOC, suggesting that the miR–181a–SFRP4 axis can be evaluated as a novel biomarker for β-catenin–targeted therapy in this disease. Significance: These results demonstrate that miR-181a is an activator of Wnt signaling that drives stemness and chemoresistance in HGSOC and may be targeted therapeutically in recurrent disease.

BackgroundCancer cell phenotypes evolve over the course of a tumor’s treatment. The phenotypes that emerge and disappear over time will be specific to each drug regimen and type of cancer. Chemotherapy remains one of the most common and effective treatments for metastatic breast cancer patients; however, resistance to chemotherapy inevitably emerges. Cancer chemotherapy treatment regimens are not designed to target emerging chemo-resistance, despite its clear importance in progressive cancer. This study focuses on finding sequential treatment strategies that target acquired chemo-resistant states and optimize response to chemotherapy.MethodsIn this study, we used heterogeneous tumor samples from patients to identify subclones resistant to chemotherapy. Using flow cytometry for stem cell markers and DNA sequencing to define subclonal population changes, we measured the enrichment of cancer stem cell-like (CSL) phenotypes in subclones that survive chemotherapy. We then analyzed breast cancer patient tumor organoids and cell line acquisition of CSL traits following chemotherapy, as well as the ability of different drugs to reverse acquired resistance, using flow cytometry, mammosphere assays, and single cell RNA-sequencing analysis.ResultsWe show that in progressive estrogen receptor positive (ER+) metastatic breast cancer patients, resistant tumor subclones that emerge following chemotherapy have increased CSL abundance. Further, in vitro organoid growth of ER+ patient cancer cells also shows that chemotherapy treatment leads to increased abundance of ALDH+/CD44+ CSL cells. Chemotherapy induced CSL abundance is blocked by treatment with a pan-HDAC inhibitor, belinostat. Further, belinostat treatment diminished both mammosphere formation and size following chemotherapy, also indicating a decrease in progenitor CSL traits. HDAC inhibitors specific to class IIa (HDAC4, HDAC5) and IIb (HDAC6) were shown to primarily reverse the chemo-resistant CSL state. Single-cell RNA sequencing analysis with patient samples showed that HDAC targets and MYC signaling were promoted by chemotherapy and inhibited upon HDAC inhibitor treatment.ConclusionThese findings indicate that HDAC inhibition can block chemotherapy-induced drug resistant phenotypes with ‘one-two punch’ strategy in refractory breast cancer cells.

No standardized curricula exist for training residents in the special needs of children with medical complexity. We assessed resident satisfaction, knowledge, and behavior after implementing a novel online curriculum composed of multimedia modules on care of children with medical complexity utilizing virtual simulation.We conducted a randomized controlled trial of residents across North America. A Web-based curriculum of 6 self-paced, interactive, multimedia modules was developed. Readings for each topic served as the control curriculum. Residents were randomized to 1 of 2 groups, each completing 3 modules and 3 sets of readings that were mutually exclusive. Outcomes included resident scores on satisfaction, knowledge-based assessments, and virtual simulation activities.Four hundred forty-two residents from 56 training programs enrolled in the curriculum, 229 of whom completed it and were included in the analysis. Subjects were more likely to report comfort with all topics if they reviewed modules compared to readings (P ≤ .01 for all 6 topics). Posttest knowledge scores were significantly higher than pretest scores overall (mean increase in score 17.7%; 95% confidence interval 16.0, 19.4), and the mean pre-post score increase for modules was significantly higher than readings (20.9% vs 15.4%, P .001). Mean scores on the verbal handoff virtual simulation increased by 1.1 points (95% confidence interval 0.2, 2.0, P = .02). There were no significant differences found in pre-post performance for the device-related emergency virtual simulation.There was high satisfaction, significant knowledge acquisition, and specific behavior change after participating in this innovative online curriculum. This is the first multisite, randomized trial assessing satisfaction, knowledge impact, and behavior change in a virtually simulated environment with pediatric trainees.

Obesity confers an independent risk for carcinogenesis. In the liver, steatosis often proceeds cancer formation; however, the mechanisms by which steatosis promotes carcinogenesis is unknown. We hypothesize that steatosis alters the microenvironment to promote proliferation of tumor initiating cells (TICs) and carcinogenesis. We used several liver cancer models to address the mechanisms underlying the role of obesity in cancer and verified these findings in patient populations. Using bioinformatics analysis and verified by biochemical assays, we identified that hepatosteatosis resulting from either Pten deletion or transgenic expression of HCV core/NS5A proteins, promotes the activation of Wnt/β-catenin. We verified that high fat diet lipid accumulation is also capable of inducing Wnt/β-catenin. Caloric restriction inhibits hepatosteatosis, reduces Wnt/β-catenin activation and blocks the expansion of TICs leading to complete inhibition of tumorigenesis without affecting the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) loss regulated protein kinase B (AKT) activation. Pharmacological inhibition or loss of the Wnt/β-catenin signal represses TIC growth in vitro, and decreases the accumulation of TICs in vivo. In human liver cancers, ontology analysis of gene set enrichment analysis (GSEA)-defined Wnt signature genes indicates that Wnt signaling is significantly induced in tumor samples compared with healthy livers. Indeed, Wnt signature genes predict 90% of tumors in a cohort of 558 patient samples. Selective depletion of macrophages leads to reduction of Wnt and suppresses tumor development, suggesting infiltrating macrophages as a key source for steatosis-induced Wnt expression. These data established Wnt/β-catenin as a novel signal produced by infiltrating macrophages induced by steatosis that promotes growth of tumor progenitor cells, underlying the increased risk of liver tumor development in obese individuals.

Background: Accurate assessment of prognostic and predictive biomarkers (estrogen receptor, progesterone receptor, HER2) plays a critical role in the clinical management of breast cancer. Triple negative breast cancers (TNBCs) lack the expression of all three targets, and no targetable molecular pathways have been identified to date. Hence, TNBCs are treated with non-targeted, cytotoxic chemotherapeutic agents (e.g. paclitaxel), and are characterized by high rates of drug resistance and metastatic relapse. CREB binding protein (CBP) has been implicated in cell growth and malignant transformation in various cancers. CBP is an important co-activator in the β-catenin driven transcription, including the Wnt signaling pathway, which has been implicated in TNBC biology. The Kahn lab has developed a specific CBP-binding small molecule inhibitor, ICG-001. We hypothesized that CBP-signaling plays an important role in TNBC biology and may provide a novel therapeutic target. Methods: We used TOP-flash assay to quantify Wnt signaling activity in TNBC. ICG-001 treatment combined with RNA Seq was used to characterize the role of CBP in TNBC cell line models. Co-immunoprecipitation (CoIP), protein and gene expression studies, as well as gene knock-down were used for validation. In vitro drug resistant cell line models as well as in vivo cell line (n=40 mice) and patient derived xenografts (PDX) in NOD scid gamma mouse models (2 patients, n=40 mice per patient) (treatmenent groups: control, paclitaxel, ICG-001, paclitaxel+ICG-001, n=5 mice per condition, primary and secondary implantation) were used to establish the effect of CBP inhibition via ICG-001 in TNBC on drug resistance and metastasis. We used the TCGA breast cancer data set to substantiate the experimental results. Results: We demonstrated that gene expression in TNBC is CBP, but not Wnt signaling dependent, and can be disrupted via ICG-001. RNA Seq analysis of TNBC cells treated with ICG-001 revealed Forkhead box M1 (FOXM1) as a potential downstream regulator. CoIP demonstrated that CBP binding to the FOXM1/β-catenin transcriptional complex. Treatment with ICG-001 revealed that CBP/FOXM1 binding, but not FOXM1/ β-catenin binding, is critical for FOXM1 expression. The PDX mouse models demonstrated that FOXM1 expression correlates with response to chemotherapy and disease recurrence in vivo. Treatment with ICG-001 sensitized FOXM1 high tumors to chemotherapeutic treatment and statistically significantly reduced tumor growth in serial transplantation experiments. Comparison of clinical data with FOXM1 expression in tumor samples from patients indicated that high levels of FOXM1 were associated with disease relapse and poor survival outcomes. Conclusion: CBP/FOXM1 binding is critical for FOXM1 expression. Targeting CBP/FOXM1 binding via ICG-001 could provide a novel therapeutic strategy in TNBC. The use of clinically annotated tissue microarrays containing a total of 430 breast tissue cores (51 TNBC cases) is currently pending to correlate nuclear protein expression of CBP and FOXM1 with survival outcomes in TNBC. FOXM1 and CBP could potentially be of value as predictive biomarkers in TNBC. These results could provide a clinical-translational rational for patient stratification based on CBP and FOXM1 expression for clinical trials exploring the therapeutic potential of FOXM1 inhibition via ICG-001 in combination with chemotherapy. Citation Format: Ring A, Nguyen C, Lenz H-J, Tripathy D, Lang JE, Kahn M. The role of CBP/FOXM1 in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-10.

Chimeric Antigen Receptor-T (CAR-T) cell immunotherapy has produced dramatic responses in hematologic malignancies. One of the challenges in the field is the lack of a simple assay for the detection of CARs on the surface of immune effector cells. In this study, we describe a novel luciferase-based assay, termed Topanga Assay, for the detection of CAR expression. The assay utilizes a recombinant fusion protein, called Topanga reagent, generated by joining the extra-cellular domain of a CAR-target in frame with one of the marine luciferases or their engineered derivatives. The assay involves incubation of CAR expressing cells with the Topanga reagent, a few washes and measurement of luminescence. The assay can detect CARs comprising either immunoglobulin- or non-immunoglobulin-based antigen binding domains. We further demonstrate that addition of epitope tags to the Topanga reagent not only allows its convenient one step purification but also extends its use for detection of CAR cells using flow cytometry. However, crude supernatant containing the secreted Topanga reagent can be directly used in both luminescence and flow-cytometry based assays without prior protein purification. Our results demonstrate that the Topanga assay is a highly sensitive, specific, convenient, economical and versatile assay for the detection of CARs.

Cancers are heterogeneous at the cellular level. Cancer stem cells/tumor initiating cells (CSC/TIC) both initiate tumorigenesis and are responsible for therapeutic resistance and disease relapse. Elimination of CSC/TIC should therefore be able to reverse therapy resistance. In principle, this could be accomplished by either targeting cancer stem cell surface markers or “stemness” pathways. Although the successful therapeutic elimination of “cancer stemness” is a critical goal, it is complex in that it should be achieved without depletion of or increases in somatic mutations in normal tissue stem cell populations. In this perspective, we will discuss the prospects for this goal via pharmacologically targeting differential Kat3 coactivator/Catenin usage, a fundamental transcriptional control mechanism in stem cell biology.

Background: Triple negative breast cancers (TNBCs) are an aggressive BC subtype, characterized by high rates of drug resistance and a high proportion of cancer stem cells (CSC). CSCs are thought to be responsible for tumor initiation and drug resistance. cAMP-response element-binding (CREB) binding protein (CREBBP or CBP) has been implicated in CSC biology and may provide a novel therapeutic target in TNBC. Methods: RNA Seq pre- and post treatment with the CBP-binding small molecule ICG-001 was used to characterize CBP-driven gene expression in TNBC cells. In vitro and in vivo TNBC models were used to determine the therapeutic effect of CBP inhibition via ICG-001. Tissue microarrays (TMAs) were used to investigate the potential of CBP and associated proteins as biomarkers in TNBC. Results: The CBP/&szlig, catenin/FOXM1 transcriptional complex drives gene expression in TNBC and is associated with increased CSC numbers, drug resistance and poor survival outcome. Targeting of CBP/&beta, catenin/FOXM1 with ICG-001 eliminated CSCs and sensitized TNBC tumors to chemotherapy. Immunohistochemistry of TMAs demonstrated a significant correlation between FOXM1 expression and TNBC subtype. Conclusion: CBP/&beta, catenin/FOXM1 transcriptional activity plays an important role in TNBC drug resistance and CSC phenotype. CBP/&beta, catenin/FOXM1 provides a molecular target for precision therapy in triple negative breast cancer and could form a rationale for potential clinical trials.

INTRODUCTION: Intubated patients with acute respiratory distress syndrome are thought to have a 5-12% incidence of barotrauma, even with protective ventilation However, little is known about the incidence of barotrauma in COVID-19 Due to high rates of observed barotrauma at this center, this retrospective cohort study aims to better characterize the incidence of barotrauma and identify predisposing factors such as inflammatory markers and disease severity indices for this high-mortality complication METHODS: Inclusion criteria were as follows: age over 18 years, positive RT-PCR for SARS-CoV2, admission to the ICU between 03/15/2020 and 06/15/2020, and a score of 5 or higher on the World Health Organization's Ordinal Scale or respiratory rate over 30 breaths per minute on admission Data were collected for the following categories developed by an internal committee of pulmonary/critical care faculty and housestaff based on similar studies: age, sex, body mass index, ferritin, d-dimer, APACHE II score, SOFA score, blood gas, ventilation mode and settings Patients with evidence of barotrauma (pneumothorax, pneumomediastinum, pneumopericardium, subcutaneous emphysema) on imaging had additional respiratory data points collected RESULTS: 78 patients met inclusion Among 38 patients who received invasive mechanical ventilation (IMV) 12 had barotrauma (32%) Of 40 patients who did not receive IMV 3 had barotrauma (8%) Of 15 cases of barotrauma, 8 had pneumothorax (2 bilateral, 6 unilateral), 9 had pneumomediastinum, 4 had pneumopericardium, 6 had subcutaneous emphysema 8 were found incidentally on imaging for non-respiratory indication Mortality in the barotrauma group was 72% for IMV & 50% for non-IMV (3 patients transferred to other hospital, 3 remain hospitalized) compared to 50% for IMV & 8% for non-IMV in patients without barotrauma Further analysis pending at submission, data to be finalized prior to presentation CONCLUSIONS: Barotrauma may be an underappreciated complication of COVID-19, perhaps serving as an independent predictor of disease severity or low lung compliance Many theories have been presented for the physiology of COVID-19 respiratory failure, but barotrauma could be evidence of or a herald sign for the low compliance phenotype

SESSION TITLE: Medical Student/Resident Disorders of the Pleura Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Barotrauma as a consequence of high alveolar pressures is well described in invasive mechanically ventilated (IMV) patients with pneumonia or pneumonitis Limited data exists on the incidence of barotrauma in patients with Coronavirus Disease 19 pneumonia (COVID-19) We present a case of barotrauma occurring in a non-intubated patient with COVID-19 receiving noninvasive positive pressure ventilation (NPPV) CASE PRESENTATION: A 43-year-old male with obesity presented with 2 days of cough and dyspnea Polymerase chain reaction (PCR) testing confirmed COVID-19 He initially required 5L of oxygen (O2) per minute by nasal cannula Due to worsening hypoxemic respiratory failure (HRF) on day 3, the patient was placed on high-flow nasal cannula (HFNC) at 90% FiO2 at a flow rate of 30 L/min By day 7, the patient had worsening HRF and increased work of breathing He was transitioned to continuous positive airway pressure (CPAP) at 100% FiO2 with continuous pressure set at 12 cm H2O On day 13, he developed tachycardia and desaturation Chest CT found gas dissecting along the axial interstitium resulting in pneumomediastinum, bilateral pneumothoraces, and extensive subcutaneous emphysema The lung parenchyma exhibited coarse ground glass opacities and early evidence of fibrotic changes Chest tubes were placed in each hemithorax with resolution of his pnemothoraces after 5 days His O2 requirements decreased, and he was transferred to a rehabilitation hospital on 10 L/min O2 by facemask on day 25 DISCUSSION: Barotrauma leading to pneumomediastinum and pneumothoraces are a well-documented complication of mechanical ventilation 1 In an effort to mitigate the morbidity and mortality associated with mechanical ventilation, many clinicians have shifted toward using NPPV in HRF in COVID-19, however NPPV may not necessarily protect patients against a complication more commonly associated with IMV 2,3 Progressive and cumulative alveolar injury may predispose COVID-19 patients to barotrauma regardless of mode of ventilation 3 CONCLUSIONS: Due to the mortality associated with IMV in COVID-19, many clinicians have shifted toward using NPPV to manage HRF However, patients receiving NPPV may still be subject to complications such as barotrauma Additionally, given our evolving understanding of COVID-19 and the pulmonary parenchymal distortion seen in many patients, a deterioration in respiratory status should prompt clinicians to search for evidence of barotrauma, even in patients receiving NPPV Reference #1: Mentzer SJ, Tsuda A, Loring SH Pleural mechanics and the pathophysiology of air leaks J Thorac Cardiovasc Surg 2018;155(5):2182-2189 Reference #2: Xu XP, Zhang XC, Hu SL, et al Noninvasive Ventilation in Acute Hypoxemic Nonhypercapnic Respiratory Failure: A Systematic Review and Meta-Analysis Crit Care Med 2017;45(7):e727-e733 Reference #3: Sun R, Liu H, Wang X Mediastinal Emphysema, Giant Bulla, and Pneumothorax Developed during the Course of COVID-19 Pneumonia Korean J Radiol 2020;21(5):541-544 DISCLOSURES: no disclosure on file for Nikhil Barot;No relevant relationships by Michael Kahn, source=Web Response No relevant relationships by Nader Kamangar, source=Web Response No relevant relationships by Jay Thetford, source=Web Response

To compare the characteristics of preterm premature rupture of membranes (PPROM) between twin and singleton pregnancies.This was a retrospective study of all women with twin and singleton pregnancies admitted with PPROM between 24-34 weeks of gestation.Overall 698 women with PPROM were eligible for the study: 101 (14.5%) twins and 597 (85.5%) singletons. Twins presented with PPROM at a more advanced gestational age compared with singletons (29.1 ± 2.7 vs. 28.5 ± 2.8 weeks, p = 0.03). The latency period was shorter in twins compared with singletons, especially for women presenting after 28 weeks of gestation (5.0 ± 0.8 vs. 7.0 ± 0.4 days, p = 0.01). Women with twins were more likely to deliver within 48 h (OR: 2.7; 95%CI: 1.7-4.2) and were less likely to deliver within 2-7 days (OR: 0.5; 95%CI: 0.3-0.9) following PPROM. The rate of clinical chorioamnionitis or placental abruption following PPROM was lower in twins compared with singletons (15.8% vs. 26.0%, p = 0.03).PPROM in twin pregnancies tends to occur at a more advanced gestational age, is associated with a shorter latency period and is less likely to be complicated by chorioamnionitis or placental abruption compared with singletons. This information may be useful for counseling and management decisions in cases of PPROM in women with twins.

Physicians play a significant role in screening for domestic violence. However, little information is available about the prevalence of physicians who experience intimate partner violence (IPV) or the implications for their clinical practice. National surveys indicate a potential prevalence of 16% for sexual abuse and 32% for abuse by an intimate partner. This extrapolates to more than 395,000 potential physician victims, the majority of which are women.We conducted a systematic review of IPV and physician victims from 1990 to 2014 that included peer-reviewed journals, trade books, and dissertations that referenced physician victims.We identified 17 publications; nine quantitative studies, four first-person accounts, one qualitative study, and a qualitative dissertation that included two physician subjects. Two case studies of victimized physicians were identified in trade books. Quantitative results noted that women reported higher prevalence for all experiences of violence [childhood exposure (6%-32%), adult IPV exposure (7%-24%)] than men (6%-10%).This review highlights the need for improved understanding of physician experience with IPV, and development of physician-sensitive resources and treatment approaches. Contributions and limitations are provided for each publication. IPV exposure impacts clinical practice, including reticence to consistently screen patients. Lower reported prevalence may be related to extreme stigma among physicians that may prevent their reporting and help seeking, but more research is needed. We provide recommendations for clinical practice, education, and future research.

Airway management in trauma is a crucial skill, because patients are at risk of aspiration, hypoxia, and hypoventilation, all of which may be fatal in the setting of increased intracranial pressure. The King Laryngeal Tube reusable supraglottic airway (King Systems, Noblesville, IN) allows for temporary management of a difficult airway but poses a challenge when an attempt is made to exchange the device for an endotracheal tube, often managed by emergency tracheostomy. We describe a novel fiberoptic, video laryngoscope-assisted approach to intubation in a difficult trauma airway with an in situ King Laryngeal Tube.

223 Background: Somatic mutations at adenomatous polyposis coli ( APC) gene, found in ~75% of colorectal cancers (CRC), are under-represented in microsatellite instable (MSI-H) tumors. While several studies have suggested worse outcomes for CRC patients (pts) with wild-type APC ( APC-WT), the prognostic implication of this genomic alteration in metastatic CRC (mCRC) is not well defined. Methods: APC prognostic value was evaluated in 331 stage IV microsatellite stable (MSS) CRC pts treated in our institution. Next-generation genomic analysis (FoundationOne) was used to characterize the molecular characteristics of APC-WT and mutant APC ( APC-MT) pts. Findings were validated on a public database of stage IV colon cancer from MSKCC. Results: APC-WT was present in 26% of mCRC patients. In comparison to APC-MT population (n = 244), APC-WT pts (n = 87) tended to be younger (median age: 49 vs. 58 years), right-sided (44% vs. 24%), BRAF-V600E mutated (25% vs. 5%), p53 WT (38% vs. 21%) and RAS WT (66% vs. 53%). APC-WT tumors were associated with other Wnt activating alterations ( CTNNB1, FBXW7, RNF43, ARID1A and SOX9). Among those, RNF43 and CTNNB1 were more significantly represented in the APC-WT vs APC-MT population (12% vs 1% and 11% vs 3%, respectively). APC-WT pts had a worse overall survival (OS) than APC-MT pts (30 vs 48 months, HR = 1.809, 95% CI 1.260-2.596, p < 0.0001). Using a multivariate model correcting for primary tumor location, RAS and BRAF status, APC-WT was predictive of poor survival (HR = 1.7, p = 0.001) in our data set. The prognostic implication of APC-WT on OS were confirmed further in a similar multivariate model of 433 stage IV pts from MSKCC public database (HR = 1.6, P = 0.01). Conclusions: APC-WT is associated with poor OS in MSS mCRC regardless of RAS, BRAF status. Compared with APC-MT mCRC tumors, APC-WT tumors were associated with other activating alterations of Wnt pathway, including RNF43 and CTNBB1.

A simple, accurate, sensitive and robust assay that can rapidly and specifically measure the death of target cells would have applications in many areas of biomedicine and particularly for the development of novel cellular- and immune-therapeutics. In this study, we describe a novel cytotoxicity assay, termed the Matador assay, which takes advantage of the extreme brightness, stability and glow-like characteristics of recently discovered novel marine luciferases and their engineered derivatives. The assay involves expression of a luciferase of interest in target cells in a manner so that it is preferentially retained within the healthy cells but is either released from dead and dying cells or whose activity can be preferentially measured in dead and dying cells. We demonstrate that this assay is highly sensitive, specific, rapid, and can be performed in a single-step manner without the need for any expensive equipment. We further validate this assay by demonstrating its ability to detect cytotoxicity induced by several cellular and immune-therapeutic agents including antibodies, natural killer cells, chimeric antigen receptor expressing T cells and a bispecific T cell engager.

PARK2 (PARKIN) is an E3 ubiquitin ligase whose dysfunction has been associated with the progression of Parkinsonism and human malignancies, and its role in cancer remains to be explored. In this study, we report that PARK2 is frequently deleted and underexpressed in human glioma, and low PARK2 expression is associated with poor survival. Restoration of PARK2 significantly inhibited glioma cell growth both in vitro and in vivo, whereas depletion of PARK2 promoted cell proliferation. PARK2 attenuated both Wnt- and EGF-stimulated pathways through downregulating the intracellular level of β-catenin and EGFR. Notably, PARK2 physically interacted with both β-catenin and EGFR. We further found that PARK2 promoted the ubiquitination of these two proteins in an E3 ligase activity-dependent manner. Finally, inspired by these newly identified tumor-suppressive functions of PARK2, we tested and proved that combination of small-molecule inhibitors targeting both Wnt–β-catenin and EGFR–AKT pathways synergistically impaired glioma cell viability. Together, our findings uncover novel cancer-associated functions of PARK2 and provide a potential therapeutic approach to treat glioma. Cancer Res; 75(9); 1815–27. ©2015 AACR.

Twin fetuses grow slower during the third trimester compared with singletons. However, the extent to which the relative smallness of twins is the result of placenta-mediated factors similar to those associated with fetal growth restriction in singletons remains unclear. Our aim was to address this question by comparing placental findings between small for gestational age (SGA) twins and SGA singletons.Retrospective cohort study of all SGA non-anomalous newborns from singleton and dichorionic twin pregnancies in a single tertiary referral center between 2002 and 2015. SGA was defined as birth weight10th percentile for gestational age according to sex-specific national reference charts. Placental findings were compared between SGA twins and SGA singletons and were classified into lesions associated with maternal vascular malperfusion, fetal vascular malperfusion, placental hemorrhage and chronic villitis.A total of 532 SGA twins and 954 SGA singletons met the inclusion criteria. SGA twins had a higher mean placental weight (371 ± 103 g vs. 319 ± 107, p 0.001) and a lower fetal-placental ratio (6.0 ± 2.5 vs. 6.7 ± 3.2, p 0.001) compared with SGA singletons. Compared with SGA singletons, SGA twins were less likely to have any placental pathology (aOR 0.37, 95%-CI 0.29-0.46), hypercoiled cord (aOR 0.45, 95%-CI 0.33-0.61), placental weight10th% (aOR 0.13, 95%-CI 0.08-0.20), maternal vascular malperfusion pathology (aOR 0.24, 95%-CI 0.18-0.30) and fetal vascular malperfusion pathology (aOR 0.62, 95%-CI 0.48-0.82). By contrast, SGA twins had higher odds of a marginal or velamentous cord insertion compared with SGA singletons (aOR 13.82, 95%-CI 10.44-18.30). Similar significant associations were observed in subgroups of SGA fetuses with a birth weight below the 5th and 3rd percentile for gestational age.Our findings illustrate that the mechanisms underlying reduced fetal growth in dichorionic twins differ from those involved in singletons, and may provide support to the hypothesis that smallness in dichorionic twins may be more benign than in singletons.

Background and objective The development of the tyrosine kinase inhibitor Imatinib (IM) represents a milestone in CML (Chronic Myeloid Leukemia) treatment. However, it is not curative and patients develop IM resistance. IM resistance has been previously correlated with the emergence of drug-resistant LIC/LSC (Leukemia Initiating Cell/Leukemia Stem Cell) and increased nuclear catenin levels and enhanced Wnt signaling. It has been demonstrated previously that drug resistant CML LIC/LSC can be safely eliminated both in vitro and in vivo via disruption of the CBP/catenin interaction, utilizing the highly biochemically selective small molecule CBP/catenin antagonist ICG- 001. Methods Here, we utilized an in vitro IM selection of primary CML patients' samples to identify drug-resistant LIC/LSC populations. In this report, we characterized the drug-resistant CML LIC/LSC population using FACS, Smartchip qPCR and colony assays to analyze cell surface markers, transcriptomics and function. Results As opposed to previous characterization of the CML leukemic stem cell population as being either CD34+CD38- or CD34+CD38+, the in vitro selected Imatinib resistant (IM-R) CML LSC population was consistently CD34-CD38-. In Long-Term Culture Initiating Cell assay (LTC-IC, a surrogate assay for long term repopulating stem cells), our results suggest that the CBP/catenin antagonist ICG- 001 sensitizes LIC/LSC to IM treatment by forced differentiative elimination of the CML LIC/LSC population. Conclusion In vitro selected IM resistant cells are negative for both CD34 and CD38 by FACS analysis. These cells acquire CD34/CD38 expression after co-culture with stromal cells. CBP/catenin antagonist ICG-001 facilitates IM function in eliminating these cells.

OBJECTIVE To explore the hypothesis that selective placental pathology affecting the nonpresenting twin is a significant contributory factor mediating the smaller size at birth of nonpresenting dichorionic twins. METHODS We conducted a retrospective cohort study of all dichorionic twin deliveries in a single tertiary center between 2002 and 2015 where by departmental policy, all placentas from multifetal gestations are routinely sent for pathologic examination. Maternal charts, neonatal charts, and pathology reports were reviewed. Placental abnormalities were classified into lesions associated with maternal vascular malperfusion, fetal vascular malperfusion, placental hemorrhage, and chronic villitis. Comparison of neonatal outcomes and placental abnormalities was made between all nonpresenting and all presenting twins as well as within twin pairs. RESULTS A total of 1,322 women with dichorionic twins were studied. Nonpresenting twins were smaller at birth compared with the presenting cotwin starting at 32 weeks of gestation (birth weight [±standard deviation] 2,224±666 g compared with 2,278±675 g, P=.036). Nonpresenting twins had smaller placentas (361±108 g compared with 492±129 g, P

WNT–β-catenin signalling is involved in a multitude of developmental processes and the maintenance of adult tissue homeostasis by regulating cell proliferation, differentiation, migration, genetic stability and apoptosis, as well as by maintaining adult stem cells in a pluripotent state. Not surprisingly, aberrant regulation of this pathway is therefore associated with a variety of diseases, including cancer, fibrosis and neurodegeneration. Despite this knowledge, therapeutic agents specifically targeting the WNT pathway have only recently entered clinical trials and none has yet been approved. This Review examines the problems and potential solutions to this vexing situation and attempts to bring them into perspective.

The Wnt signaling pathway controls several cell processes, such as motility and proliferation during embryonic development. Wnt signaling is also involved in the maintenance of potency and the induction of differentiation in stem cells. Aberrant Wnt signaling is implicated in several cancer types. Particularly in colorectal cancer (CRC), the Wnt-β-catenin signaling cascade is at the center of the carcinogenesis, and mutations in this pathway can be found in almost all CRC patients. We discuss the potential of targeting Wnt-β-catenin signaling with a brief overview of the pathway and the most promising pathway inhibitors.

Background: While several assays currently exist to enumerate circulating tumor cells (CTCs), limitations to existing assays include reliance on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM) and difficulty isolating pure populations of rare CTC RNA from peripheral blood without background subtraction of leukocytes. We evaluated the biomarkers CD146 and CD49f as a strategy to isolate CTCs that underwent epithelial to mesenchymal transition (EMT). We hypothesized that using immunomagnetic separation and fluorescence activated cell sorting (IE/FACS) it is possible to isolate RNA derived from EpCAM-negative CTCs. Methods: 10 breast cancer cell lines were acquired from the ATCC, authenticated by short tandem repeat profiling, and stratified according to subtype: HER2 positive (SKBR3, MDA-MB-453, SUM190), luminal A (T47D, MCF7, ZR751), luminal B (BT474) and basal-like (SUM149, MDA-MB-231, Hs578T). IE/FACS with EMT gates was performed using CD146 ferrofluid particles via a magnetic separator followed by incubation with Thioflavin T, CD 31 and CD45 PE-Cy7 (for human or mouse blood), CD146-V450, and CD49f-A647. FACS sorting was performed using a FACS Aria II (BD Biosciences). Known quantities of cells were spiked into PBS or peripheral blood (PB) from healthy female donors and subjected to IE/FACS with EMT gates. Absolute cell counts and recovery rates were determined using the TruCOUNT method (BD Biosciences) with acquisition of 35,000 beads. MDA-MB-231 xenografts were established in 4 male NOD/SCID/IL2 gamma chain deficient mice; cardiac punctures were performed to acquire a complete xenograft PB volume for IE/FACS. Sorted xenograft CTCs, human PB and MDA-MB-231 were used to isolate RNA, amplify and profile via Affymetrix human gene ST 1.0 arrays. The effect of sample processing with IE/FACS was assessed using our EpCAM gates (EpCAM ferrofluid, EpCAM-PE, thioflavin, CD45 PE-Cy7) to sort BT474 and profiled via next generation sequencing (NGS) using the Helicos Single Molecule sequencing approach. Results: MDA-MB-231 spiked into PB from an unaffected mouse and negative mouse PB controls were used to establish gating thresholds. This gating strategy recovered 14% of MDA-MB-231 cells from spiked human PB via the TruCOUNT method, which represented a 200% yield versus EpCAM based IE/FACS. CTCs were successfully isolated from 4/4 mice bearing xenografts (ranging from 193-6352 cells captured per ∼1mL mouse blood). Principal component analysis demonstrated that gene expression may differentiate xenograft CTCs from PB and MDA-MB-231. Recovery rates using the EMT gates for 10 cell lines spiked into PBS and human PB will be reported and compared to our typical EpCAM gating strategy. Pearson's correlation coefficient of BT474 spiked into PB and recovered via IE/FACS using an EpCAM gating strategy was 92% by NGS. Conclusions: We have established a method for the isolation and expression profiling of EpCAM-negative CTCs as well as EpCAM-positive CTCs. Future studies are required to determine if expression profiling of CTCs can be used as prognostic, predictive and discovery tools. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-07.

Cancer initiation and progression have been attributed to newly discovered subpopulations of self-renewing, highly tumorigenic, drug-resistant tumor cells termed cancer stem cells. Recently, we and others reported a new phenotypic plasticity wherein highly tumorigenic, drug-resistant cell populations could arise not only from pre-existing cancer stem-like populations but also from cancer cells lacking these properties. In the current study, we hypothesized that this newfound phenotypic plasticity may be mediated by PI3K/Akt and Wnt/β-catenin signaling, pathways previously implicated in carcinogenesis, pluripotency and drug resistance. Using GFP expression, Hoechst dye exclusion and fluorescence activated cell sorting (FACS) of cancer cell lines, we identified and tracked cancer stem-like side populations (SP) of cancer cells characterized by high tumorigenicity and drug resistance. We found that pharmacological inhibition or genetic depletion of PI3K and AKT markedly reduced the spontaneous conversion of nonside population (NSP) cells into cancer stem-like SP cells, whereas PI3K/Akt activation conversely enhanced NSP to SP conversion. PI3K/AKT signaling was mediated through downstream phosphorylation of GSK3β, which led to activation and accumulation of β-catenin. Accordingly, pharmacological or genetic perturbation of GSK3β or β-catenin dramatically impacted conversion of NSP to SP. Further downstream, β-catenin's effects on NSP-SP equilibrium were dependent upon its interaction with CBP, a KAT3 family coactivator. These studies provide a mechanistic model wherein PI3K/Akt/β-catenin/CBP signaling mediates phenotypic plasticity in and out of a drug-resistant, highly tumorigenic state. Therefore, targeting this pathway has unique potential for overcoming the therapy resistance and disease progression attributed to the cancer stem-like phenotype.

Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia; however, little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CREB-binding protein (CBP)) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300 leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small-molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1–110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/β- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using chromatin immunoprecipitation assay, we demonstrate occupancy of the survivin promoter by CBP that is decreased by ICG-001 in primary ALL. CBP mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.

Repair of the lung epithelium after injury is integral to the pathogenesis and outcomes of diverse inflammatory lung diseases. We previously reported that β-catenin signaling promotes epithelial repair after inflammatory injury, but the β-catenin target genes that mediate this effect are unknown. Herein, we examined which β-catenin transcriptional coactivators and target genes promote epithelial repair after inflammatory injury. Transmigration of human neutrophils across cultured monolayers of human lung epithelial cells resulted in a fall in transepithelial resistance and the formation of discrete areas of epithelial denudation (“microinjury”), which repaired via cell spreading by 96 h. In mice treated with intratracheal (i.t.) LPS or keratinocyte chemokine, neutrophil emigration was associated with increased permeability of the lung epithelium, as determined by increased bronchoalveolar lavage (BAL) fluid albumin concentration, which decreased over 3–6 days. Activation of β-catenin/p300-dependent gene expression using the compound ICG-001 accelerated epithelial repair in vitro and in murine models. Neutrophil transmigration induced epithelial expression of the β-catenin/p300 target genes Wnt-induced secreted protein (WISP) 1 and cysteine-rich (Cyr) 61, as determined by real-time PCR (qPCR) and immunostaining. Purified neutrophil elastase induced WISP1 upregulation in lung epithelial cells, as determined by qPCR. WISP1 expression increased in murine lungs after i.t. LPS, as determined by ELISA of the BAL fluid and qPCR of whole lung extracts. Finally, recombinant WISP1 and Cyr61 accelerated repair, and Cyr61-neutralizing antibodies delayed repair of the injured epithelium in vitro. We conclude that β-catenin/p300-dependent expression of WISP1 and Cyr61 is critical for epithelial repair and represents a potential therapeutic target to promote epithelial repair after inflammatory injury.

Summary Transforming growth factor β1 (TGF-β1) is known to be both anti-inflammatory and profibrotic. Cross-talk between TGF-β/Smad and Wnt/β-catenin pathways in epithelial–mesenchymal transition (EMT) suggests a specific role for β-catenin in profibrotic effects of TGF-β1. However, no such mechanistic role has been demonstrated for β-catenin in the anti-inflammatory effects of TGF-β1. In the present study, we explored the role of β-catenin in the profibrotic and anti-inflammatory effects of TGF-β1 by using a cytosolic, but not membrane, β-catenin knockdown chimera (F-TrCP-Ecad) and the β-catenin/CBP inhibitor ICG-001. TGF-β1 induced nuclear Smad3/β-catenin complex, but not β-catenin/LEF-1 complex or TOP-flash activity, during EMT of C1.1 (renal tubular epithelial) cells. F-TrCP-Ecad selectively degraded TGF-β1-induced cytoplasmic β-catenin and blocked EMT of C1.1 cells. Both F-TrCP-Ecad and ICG-001 blocked TGF-β1-induced Smad3/β-catenin and Smad reporter activity in C1.1 cells, suggesting that TGF-β1-induced EMT depends on β-catenin binding to Smad3, but not LEF-1 downstream of Smad3, through canonical Wnt. In contrast, in J774 macrophages, the β-catenin level was low and was not changed by interferon-γ (IFN-γ) or lipopolysaccharide (LPS) with or without TGF-β1. TGF-β1 inhibition of LPS-induced TNF-α and IFN-γ-stimulated inducible NO synthase (iNOS) expression was not affected by F-TrCP-Ecad, ICG-001 or by overexpression of wild-type β-catenin in J774 cells. Inhibition of β-catenin by either F-TrCP-Ecad or ICG-001 abolished LiCl-induced TOP-flash, but not TGF-β1-induced Smad reporter, activity in J774 cells. These results demonstrate for the first time that β-catenin is required as a co-factor of Smad in TGF-β1-induced EMT of C1.1 epithelial cells, but not in TGF-β1 inhibition of macrophage activation. Targeting β-catenin may dissociate the TGF-β1 profibrotic and anti-inflammatory effects.

Background Hepatic fibrosis is a prominent pathological feature associated with chronic liver disease including non-alcoholic hepatosteatosis (NASH), and a precursor for liver cancer development. We previously reported that PTEN loss in the liver, which leads to hyperactivated liver insulin signaling results in NASH development. Here we used the same mouse model to study the progression from steatosis to fibrosis. Results The Pten null livers develop progressive liver fibrosis as indicated by Sirius Red staining and increased expression of collagen I, Timp 1, SMAα, and p75NTR. Consistently, hepatic stellate cells (HSCs) isolated from Pten null livers are readily activated when compared with that from mice with intact PTEN. Deletion of AKT2, the downstream target of PTEN signal, blocked NASH development, and alleviated fibrosis. HSCs from the Pten/Akt2 double null mice are quiescent like those isolated from the control livers. Our analysis shows that the activation of HSCs does not depend on the intrinsic signals regulated by PI3K/AKT, the target of PTEN, but does depend on steatosis and injury to the liver. During the progression of liver fibrosis in the Pten null model, Wnt ligands and signaling receptor are induced, concurrent with the reduction of sFRP5, a Wnt antagonist. We showed that treatment of HSCs with Wnt receptor antagonist blocks the observed morphological changes when HSCs undergo activation in culture. This signal appears to be mediated by β-catenin, as manipulating β-catenin signaling alters marker gene expressions of HSC activation. Conclusions Wnt/β-catenin activation serves as an important mediator for fibrosis development resulting from NASH using a mouse model where NASH is mimicked by PTEN loss.

Recent studies suggest CD133, a surface protein widely used for isolation of colon cancer stem cells, to be associated with tumor angiogenesis and recurrence. We hypothesized that gene expression levels and germline variations in CD133 will predict clinical outcome in patients with metastatic colorectal cancer (mCRC), treated in first-line setting with 5-fluorouracil, oxaliplatin and bevacizumab (BV), and we investigated whether there is a correlation with gene expression levels of CD133, vascular endothelial growth factor (VEGF) and its receptors. We evaluated intra-tumoral gene expression levels by quantitative real-time (RT) PCR from 54 patients and three germline variants of the CD133 gene by PCR-restriction-fragment length polymorphism from 91 patients with genomic DNA. High gene expression levels of CD133 (>7.76) conferred a significantly greater tumor response (RR=86%) than patients with low expression levels (7.76, RR=38%, adjusted P=0.003), independent of VEGF or its receptor gene expression levels. Gene expression levels of CD133 were significantly associated with VEGF and its receptors messenger RNA levels (VEGFR-1 (P

Cancer stem cells (CSCs), also known as tumor initiating cells are now considered to be the root cause of most if not all cancers, evading treatment and giving rise to disease relapse. They have become a central focus in new drug development. Prospective identification, understanding the key pathways that maintain CSCs, and being able to target CSCs, particularly if the normal stem cell population could be spared, could offer an incredible therapeutic advantage. The Wnt signaling cascade is critically important in stem cell biology, both in homeostatic maintenance of tissues and organs through their respective somatic stem cells and in the CSC/tumor initiating cell population. Aberrant Wnt signaling is associated with a wide array of tumor types. Therefore, the ability to safely target the Wnt signaling pathway offers enormous promise to target CSCs. However, just like the sword of Damocles, significant risks and concerns regarding targeting such a critical pathway in normal stem cell maintenance and tissue homeostasis remain ever present. With this in mind, we review recent efforts in modulating the Wnt signaling cascade and critically analyze therapeutic approaches at various stages of development.

The Wnt signaling pathway is critically involved in both the development and homeostasis of tissues via regulation of their endogenous stem cells. Aberrant Wnt signaling has been described as a key player in the initiation of and/or maintenance and development of many cancers, via affecting the behavior of Cancer Stem Cells (CSCs). CSCs are considered by most to be responsible for establishment of the tumor and also for disease relapse, as they possess inherent drug-resistance properties. The development of new therapeutic compounds targeting the Wnt signaling pathway promises new hope to eliminate CSCs and achieve cancer eradication. However, a major challenge resides in developing a strategy efficient enough to target the dysregulated Wnt pathway in CSCs, while being safe enough to not damage the normal somatic stem cell population required for tissue homeostasis and repair. Here we review recent therapeutic approaches to target the Wnt pathway and their clinical applications.

Injury to the epithelium is integral to the pathogenesis of many inflammatory lung diseases, and epithelial repair is a critical determinant of clinical outcome. However, the signaling pathways regulating such repair are incompletely understood. We used in vitro and in vivo models to define these pathways. Human neutrophils were induced to transmigrate across monolayers of human lung epithelial cells in the physiological basolateral-to-apical direction. This allowed study of the neutrophil contribution not only to the initial epithelial injury, but also to its repair, as manifested by restoration of transepithelial resistance and reepithelialization of the denuded epithelium. Microarray analysis of epithelial gene expression revealed that neutrophil transmigration activated β-catenin signaling, and this was verified by real-time PCR, nuclear translocation of β-catenin, and TOPFlash reporter activity. Leukocyte elastase, likely via cleavage of E-cadherin, was required for activation of β-catenin signaling in response to neutrophil transmigration. Knockdown of β-catenin using shRNA delayed epithelial repair. In mice treated with intratracheal LPS or keratinocyte chemokine, neutrophil emigration resulted in activation of β-catenin signaling in alveolar type II epithelial cells, as demonstrated by cyclin D1 expression and/or reporter activity in TOPGAL mice. Attenuation of β-catenin signaling by IQ-1 inhibited alveolar type II epithelial cell proliferation in response to neutrophil migration induced by intratracheal keratinocyte chemokine. We conclude that β-catenin signaling is activated in lung epithelial cells during neutrophil transmigration, likely via elastase-mediated cleavage of E-cadherin, and regulates epithelial repair. This pathway represents a potential therapeutic target to accelerate physiological recovery in inflammatory lung diseases.

Relapse of drug-resistant acute lymphoblastic leukemia (ALL) has been associated with increased expression of survivin/BIRC5, an inhibitor of apoptosis protein, suggesting a survival advantage for ALL cells. In the present study, we report that inhibition of survivin in patient-derived ALL can eradicate leukemia. Targeting survivin with shRNA in combination with chemotherapy resulted in no detectable minimal residual disease in a xenograft model of primary ALL. Similarly, pharmacologic knock-down of survivin using EZN-3042, a novel locked nucleic acid antisense oligonucleotide, in combination with chemotherapy eliminated drug-resistant ALL cells. These findings show the importance of survivin expression in drug resistance and demonstrate that survivin inhibition may represent a powerful approach to overcoming drug resistance and preventing relapse in patients with ALL.