Nuclear epidermal growth factor receptor as a therapeutic target.

Epidermal growth factor receptor (EGFR) is one of the most well-studied oncogenes with roles in proliferation, growth, metastasis, and therapeutic resistance. This intense study has led to the development of a range of targeted therapeutics including small-molecule tyrosine kinase inhibitors (TKIs), monoclonal antibodies, and nanobodies. These drugs are excellent at blocking the activation and kinase function of wild-type EGFR (wtEGFR) and several common EGFR mutants. These drugs have significantly improved outcomes for patients with cancers including head and neck, glioblastoma, colorectal, and non-small cell lung cancer (NSCLC). However, therapeutic resistance is often seen, resulting from acquired mutations or activation of compensatory signaling pathways. Additionally, these therapies are ineffective in tumors where EGFR is found predominantly in the nucleus, as can be found in triple negative breast cancer (TNBC). In TNBC, EGFR is subjected to alternative trafficking which drives the nuclear localization of the receptor. In the nucleus, EGFR interacts with several proteins to activate transcription, DNA repair, migration, and chemoresistance. Nuclear EGFR (nEGFR) correlates with metastatic disease and worse patient prognosis yet targeting its nuclear localization has proved difficult. This review provides an overview of current EGFR-targeted therapies and novel peptide-based therapies that block nEGFR, as well as their clinical applications and potential for use in oncology.

Response to high dose ipilimumab plus temozolomide after progression on standard or low dose ipilimumab in advanced melanoma: a retrospective analysis.

Background: Despite advancements in checkpoint inhibitor-based immunotherapy, patients with advanced melanoma who have progressed on standard dose ipilimumab (Ipi) + nivolumab continue to have poor prognosis. Several studies support a dose-response activity of Ipi, and one promising combination is Ipi 10mg/kg (Ipi10) + temozolomide (TMZ). Methods: We performed a retrospective cohort analysis of patients with advanced melanoma treated with Ipi10+TMZ in the immunotherapy refractory/resistant setting (n = 6), using similar patients treated with Ipi3+TMZ (n = 6) as comparison. Molecular profiling by whole exome sequencing (WES) and RNA-seq of tumors harvested through one responder's treatment was performed. Results: With a median follow up of 119 days, patients treated with Ipi10+TMZ had statistically significant longer median progression free survival of 144.5 days (range 27-219) vs 44 (26-75) in Ipi3+TMZ, p=0.04, and a trend for longer median overall survival of 154.5 days (27-537) vs 89.5 (26-548). All patients in the Ipi10 cohort had progressed on prior Ipi+Nivo. WES revealed only 12 shared somatic mutations including BRAF V600E. RNA-seq showed enrichment of inflammatory signatures, including interferon responses in metastatic lesions after standard dose Ipi + nivo and Ipi10 + TMZ compared to the primary tumor, and downregulated negative immune regulators including Wnt and TGFb signaling. Conclusion: Ipi10+TMZ demonstrated efficacy including dramatic responses in patients with advanced melanoma refractory to prior Ipi + anti-PD1, even with CNS metastases. Molecular data suggest a potential threshold of Ipi dose for activation of sufficient anti-tumor immune response, and higher dose Ipi is required for some patients.

Predicting severe chronic obstructive pulmonary disease exacerbations using quantitative CT: a retrospective model development and external validation study.

BACKGROUND: Quantitative CT is becoming increasingly common for the characterisation of lung disease; however, its added potential as a clinical tool for predicting severe exacerbations remains understudied. We aimed to develop and validate quantitative CT-based models for predicting severe chronic obstructive pulmonary disease (COPD) exacerbations. METHODS: We analysed the Subpopulations and Intermediate Outcome Measures In COPD Study (SPIROMICS) cohort, a multicentre study done at 12 clinical sites across the USA, of individuals aged 40-80 years from four strata: individuals who never smoked, individuals who smoked but had normal spirometry, individuals who smoked and had mild to moderate COPD, and individuals who smoked and had severe COPD. We used 3-year follow-up data to develop logistic regression classifiers for predicting severe exacerbations. Predictors included age, sex, race, BMI, pulmonary function, exacerbation history, smoking status, respiratory quality of life, and CT-based measures of density gradient texture and airway structure. We externally validated our models in a subset from the Genetic Epidemiology of COPD (COPDGene) cohort. Discriminative model performance was assessed using the area under the receiver operating characteristic curve (AUC), which was also compared with other predictors, including exacerbation history and the BMI, airflow obstruction, dyspnoea, and exercise capacity (BODE) index. We evaluated model calibration using calibration plots and Brier scores. FINDINGS: Participants in SPIROMICS were enrolled between Nov 12, 2010, and July 31, 2015. Participants in COPDGene were enrolled between Jan 10, 2008, and April 15, 2011. We included 1956 participants from the SPIROMICS cohort who had complete 3-year follow-up data: the mean age of the cohort was 63·1 years (SD 9·2) and 1017 (52%) were men and 939 (48%) were women. Among the 1956 participants, 434 (22%) had a history of at least one severe exacerbation. For the CT-based models, the AUC was 0·854 (95% CI 0·852-0·855) for at least one severe exacerbation within 3 years and 0·931 (0·930-0·933) for consistent exacerbations (defined as ≥1 acute episode in each of the 3 years). Models were well calibrated with low Brier scores (0·121 for at least one severe exacerbation; 0·039 for consistent exacerbations). For the prediction of at least one severe event during 3-year follow-up, AUCs were significantly higher with CT biomarkers (0·854 [0·852-0·855]) than exacerbation history (0·823 [0·822-0·825]) and BODE index 0·812 [0·811-0·814]). 6965 participants were included in the external validation cohort, with a mean age of 60·5 years (SD 8·9). In this cohort, AUC for at least one severe exacerbation was 0·768 (0·767-0·769; Brier score 0·088). INTERPRETATION: CT-based prediction models can be used for identification of patients with COPD who are at high risk of severe exacerbations. The newly identified CT biomarkers could potentially enable investigation into underlying disease mechanisms responsible for exacerbations. FUNDING: National Institutes of Health and the National Heart, Lung, and Blood Institute.

Sorting nexin-dependent therapeutic targeting of oncogenic epidermal growth factor receptor.

Overexpression and/or overactivation of the Epidermal Growth Factor Receptor (EGFR) is oncogenic in several tumor types yet targeting the kinase domain of wildtype EGFR has had limited success. EGFR has numerous kinase-independent roles, one of which is accomplished through the Sorting Nexin-dependent retrotranslocation of EGFR to the nucleus, which is observed in some metastatic cancers and therapeutically resistant disease. Here, we have utilized the BAR domain of Sorting Nexin 1 to create a peptide-based therapeutic (cSNX1.3) that promotes cell death in EGFR-expressing cancer. We evaluated the efficacy of cSNX1.3 in tumor-bearing WAP-TGFα transgenic mice (an EGFR-dependent model of breast cancer), where cSNX1.3 treatment resulted in significant tumor regression without observable toxicity. Evaluation of remaining tumor tissues found evidence of increased PARP cleavage, suggesting apoptotic tumor cell death. To evaluate the mechanism of action for cSNX1.3, we found that cSNX1.3 binds the C-terminus of the EGFR kinase domain at an interface site opposite the ATP binding domain with a Kd of ~4.0 µM. In vitro analysis found that cSNX1.3 inhibits the nuclear localization of EGFR. To determine specificity, we evaluated cancer cell lines expressing wildtype EGFR (MDA-MB-468, BT20 and A549), mutant EGFR (H1975) and non-transformed lines (CHO and MCF10A). Only transformed lines expressing wildtype EGFR responded to cSNX1.3, while mutant EGFR and normal cells responded better to an EGFR kinase inhibitor. Phenotypically, cSNX1.3 inhibits EGF-, NRG-, and HGF-dependent migration, but not HA-dependent migration. Together, these data indicate that targeting retrotranslocation of EGFR may be a potent therapeutic for RTK-active cancer.

Feasibility and Clinical Utility of a Workflow Interfacing Radiation Oncology Lung Stereotactic Body Radiation Therapy Treatment Planning and Diagnostic Radiology.

Stereotactic body radiation therapy (SBRT) is commonly used to treat early-stage non-small cell lung cancer. Beam arrangements for SBRT include multiple entry and exit pathways resulting in irregular low-dose distributions within normal lung parenchyma. An improved understanding of posttreatment radiographic changes may improve the ability to predict clinical complications including radiation pneumonitis as well as assist in early detection of local failures. Radiation treatment planning is conducted using software systems separate from diagnostic radiology, often not accessible to the diagnostic radiologist. We developed a workflow for interfacing radiation dose information from lung SBRT treatments with a diagnostic radiology picture archiving and communication system (PACS). In an anonymized PACS study folder, SBRT dose maps depicting high-dose, low-dose, and nonirradiated lung volumes were viewable side by side with pretreatment and follow-up diagnostic computed tomography scans. Clinical utility was evaluated by 2 thoracic diagnostic radiologists reviewing posttreatment diagnostic follow-up scans in the PACS both with and without radiation dose maps available. The addition of the biologically effective dose map did not significantly change identification rates of radiation induced lung injury) (92% vs 95%; P = .32) but did significantly decrease radiologic suspicion for local recurrence (22% vs 8%; P = .003). The addition of biologically effective dose maps significantly increased confidence in identifying radiation induced lung injury (7.75 vs 8.82; P = .004) and local recurrence (5.5 vs 6.6; P = .005). The recommendation for additional workup was not significantly different (10% vs 7%; P = .41). We demonstrated the feasibility and clinical utility of a workflow generating simplified radiation dose maps that are viewable within a PACS for diagnostic radiology review.

PLOSL: Population learning followed by one shot learning pulmonary image registration using tissue volume preserving and vesselness constraints.

This paper presents the Population Learning followed by One Shot Learning (PLOSL) pulmonary image registration method. PLOSL is a fast unsupervised learning-based framework for 3D-CT pulmonary image registration algorithm based on combining population learning (PL) and one-shot learning (OSL). The PLOSL image registration has the advantages of the PL and OSL approaches while reducing their respective drawbacks. The advantages of PLOSL include improved performance over PL, substantially reducing OSL training time and reducing the likelihood of OSL getting stuck in local minima. PLOSL pulmonary image registration uses tissue volume preserving and vesselness constraints for registration of inspiration-to-expiration and expiration-to-inspiration pulmonary CT images. A coarse-to-fine convolution encoder-decoder CNN architecture is used to register large and small shape features. During training, the sum of squared tissue volume difference (SSTVD) compensates for intensity differences between inspiration and expiration computed tomography (CT) images and the sum of squared vesselness measure difference (SSVMD) helps match the lung vessel tree. Results show that the PLOSL (SSTVD+SSVMD) algorithm achieved subvoxel landmark error while preserving pulmonary topology on the SPIROMICS data set, the public DIR-LAB COPDGene and 4DCT data sets.

Application of Microfluidic Systems for Breast Cancer Research.

Cancer is a disease in which cells in the body grow out of control; breast cancer is the most common cancer in women in the United States. Due to early screening and advancements in therapeutic interventions, deaths from breast cancer have declined over time, although breast cancer remains the second leading cause of cancer death among women. Most deaths are due to metastasis, as cancer cells from the primary tumor in the breast form secondary tumors in remote sites in distant organs. Over many years, the basic biological mechanisms of breast cancer initiation and progression, as well as the subsequent metastatic cascade, have been studied using cell cultures and animal models. These models, although extremely useful for delineating cellular mechanisms, are poor predictors of physiological responses, primarily due to lack of proper microenvironments. In the last decade, microfluidics has emerged as a technology that could lead to a paradigm shift in breast cancer research. With the introduction of the organ-on-a-chip concept, microfluidic-based systems have been developed to reconstitute the dominant functions of several organs. These systems enable the construction of 3D cellular co-cultures mimicking in vivo tissue-level microenvironments, including that of breast cancer. Several reviews have been presented focusing on breast cancer formation, growth and metastasis, including invasion, intravasation, and extravasation. In this review, realizing that breast cancer can recur decades following post-treatment disease-free survival, we expand the discussion to account for microfluidic applications in the important areas of breast cancer detection, dormancy, and therapeutic development. It appears that, in the future, the role of microfluidics will only increase in the effort to eradicate breast cancer.

Forced Expiratory Flow at 25%-75% Links COPD Physiology to Emphysema and Disease Severity in the SPIROMICS Cohort.

BACKGROUND: Forced expiratory volume in 1 second (FEV1) is central to the diagnosis of chronic obstructive pulmonary disease (COPD) but is imprecise in classifying disease burden. We examined the potential of the maximal mid-expiratory flow rate (forced expiratory flow rate between 25% and 75% [FEF25%-75%]) as an additional tool for characterizing pathophysiology in COPD. OBJECTIVE: To determine whether FEF25%-75% helps predict clinical and radiographic abnormalities in COPD. STUDY DESIGN AND METHODS: The SubPopulations and InteRediate Outcome Measures In COPD Study (SPIROMICS) enrolled a prospective cohort of 2978 nonsmokers and ever-smokers, with and without COPD, to identify phenotypes and intermediate markers of disease progression. We used baseline data from 2771 ever-smokers from the SPIROMICS cohort to identify associations between percent predicted FEF25%-75% (%predFEF25%-75%) and both clinical markers and computed tomography (CT) findings of smoking-related lung disease. RESULTS: Lower %predFEF25-75% was associated with more severe disease, manifested radiographically by increased functional small airways disease, emphysema (most notably with homogeneous distribution), CT-measured residual volume, total lung capacity (TLC), and airway wall thickness, and clinically by increased symptoms, decreased 6-minute walk distance, and increased bronchodilator responsiveness (BDR). A lower %predFEF25-75% remained significantly associated with increased emphysema, functional small airways disease, TLC, and BDR after adjustment for FEV1 or forced vital capacity (FVC). INTERPRETATION: The %predFEF25-75% provides additional information about disease manifestation beyond FEV1. These associations may reflect loss of elastic recoil and air trapping from emphysema and intrinsic small airways disease. Thus, %predFEF25-75% helps link the anatomic pathology and deranged physiology of COPD.

Cxcl10 Chemokine Induces Migration of ING4-Deficient Breast Cancer Cells via a Novel Cross Talk Mechanism between the Cxcr3 and Egfr Receptors.

The chemokine Cxcl10 has been associated with poor prognosis in breast cancer, but the mechanism is not well understood. Our previous study has shown that CXCL10 was repressed by the ING4 tumor suppressor, suggesting a potential inverse functional relationship. We thus investigated a role for Cxcl10 in the context of ING4 deficiencies in breast cancer. We first analyzed public gene expression data sets and found that patients with CXCL10-high/ING4-low expressing tumors had significantly reduced disease-free survival in breast cancer. In vitro, Cxcl10 induced migration of ING4-deleted breast cancer cells but not of ING4-intact cells. Using inhibitors, we found that Cxcl10-induced migration of ING4-deleted cells required Cxcr3, Egfr, and the Gßγ subunits downstream of Cxcr3 but not Gαi. Immunofluorescent imaging showed that Cxcl10 induced early transient colocalization between Cxcr3 and Egfr in both ING4-intact and ING4-deleted cells, which recurred only in ING4-deleted cells. A peptide agent that binds to the internal juxtamembrane domain of Egfr inhibited Cxcr3/Egfr colocalization and cell migration. Taken together, these results presented a novel mechanism of Cxcl10 that elicits migration of ING4-deleted cells, in part by inducing a physical or proximal association between Cxcr3 and Egfr and signaling downstream via Gßγ. These results further indicated that ING4 plays a critical role in the regulation of Cxcl10 signaling that enables breast cancer progression.

Mucus Plugs and Emphysema in the Pathophysiology of Airflow Obstruction and Hypoxemia in Smokers.

Rationale: The relative roles of mucus plugs and emphysema in mechanisms of airflow limitation and hypoxemia in smokers with chronic obstructive pulmonary disease (COPD) are uncertain.Objectives: To relate image-based measures of mucus plugs and emphysema to measures of airflow obstruction and oxygenation in patients with COPD.Methods: We analyzed computed tomographic (CT) lung images and lung function in participants in the Subpopulations and Intermediate Outcome Measures in COPD Study. Radiologists scored mucus plugs on CT lung images, and imaging software automatically quantified emphysema percentage. Unadjusted and adjusted relationships between mucus plug score, emphysema percentage, and lung function were determined using regression.Measurements and Main Results: Among 400 smokers, 229 (57%) had mucus plugs and 207 (52%) had emphysema, and subgroups could be identified with mucus-dominant and emphysema-dominant disease. Only 33% of smokers with high mucus plug scores had mucus symptoms. Mucus plug score and emphysema percentage were independently associated with lower values for FEV1 and peripheral oxygen saturation (P < 0.001). The relationships between mucus plug score and lung function outcomes were strongest in smokers with limited emphysema (P < 0.001). Compared with smokers with low mucus plug scores, those with high scores had worse COPD Assessment Test scores (17.4 ± 7.7 vs. 14.4 ± 13.3), more frequent annual exacerbations (0.75 ± 1.1 vs. 0.43 ± 0.85), and shorter 6-minute-walk distance (329 ± 115 vs. 392 ± 117 m) (P < 0.001).Conclusions: Symptomatically silent mucus plugs are highly prevalent in smokers and independently associate with lung function outcomes. These data provide rationale for targeting patients with mucus-high/emphysema-low COPD in clinical trials of mucoactive treatments.Clinical trial registered with www.clinicaltrials.gov (NCT01969344).

Clinical, Bronchoscopic, and Imaging Findings of e-Cigarette, or Vaping, Product Use-Associated Lung Injury Among Patients Treated at an Academic Medical Center.

Importance: e-Cigarette, or vaping, product use-associated lung injury (EVALI) has caused more than 2800 illnesses and 68 deaths in the United States. Better characterization of this novel illness is needed to inform diagnosis and management. Objective: To describe the clinical features, bronchoscopic findings, imaging patterns, and outcomes of EVALI. Design, Setting, and Participants: This case series of 31 adult patients diagnosed with EVALI between June 24 and December 10, 2019, took place at an academic medical center in Salt Lake City, Utah. Exposures: e-Cigarette use, also known as vaping. Main Outcomes and Measures: Symptoms, laboratory findings, bronchoscopic results, imaging patterns, and clinical outcomes. Results: Data from 31 patients (median [interquartile range] age, 24 [21-31] years) were included in the study. Patients were primarily men (24 [77%]) and White individuals (27 [87%]) who used e-cigarette products containing tetrahydrocannabinol (THC) (29 [94%]). Patients presented with respiratory (30 [97%]), constitutional (28 [90%]), and gastrointestinal (28 [90%]) symptoms. Serum inflammatory markers were elevated in all patients. Bronchoscopy was performed in 23 of 28 inpatients (82%) and bronchoalveolar lavage (BAL) revealed the presence of lipid-laden macrophages (LLMs) in 22 of 24 cases (91%). BAL samples tested positive for Pneumocystis jirovecii (3 patients [13%]), rhinovirus (2 patients [8%]), human metapneumovirus and Aspergillus (1 patient each [4%]); all except human metapneumovirus were determined to be false-positives or clinically inconsequential. The exclusive or dominant computed tomography (CT) pattern was organizing pneumonia in 23 of 26 cases (89%). Patients received antibiotics (26 [84%]) and corticosteroids (24 [77%]), and all survived; 20 patients (65%) seen in follow-up showed marked improvement, but residual symptoms (13 [65%]), radiographic opacities (8 [40%]), and abnormal pulmonary function tests (8 of 18 [44%]) were common. Conclusions and Relevance: In this case series, patients with EVALI characteristically presented with a flu-like illness with elevated inflammatory markers, LLMs on BAL samples, and an organizing pneumonia pattern on CT imaging. Bronchoscopic testing for infection had a high incidence of false-positive results. Patients had substantial residual abnormal results at early follow-up. These data suggest a limited role for bronchoscopy in typical presentations of EVALI without risk factors for alternative diagnoses and the need for careful longitudinal follow-up.

Stapled EGFR peptide reduces inflammatory breast cancer and inhibits additional HER-driven models of cancer.

BACKGROUND: The human epidermal growth factor receptor (HER) family of transmembrane tyrosine kinases is overexpressed and correlates with poor prognosis and decreased survival in many cancers. The receptor family has been therapeutically targeted, yet tyrosine kinase inhibitors (TKIs) do not inhibit kinase-independent functions and antibody-based targeting does not affect internalized receptors. We have previously demonstrated that a peptide mimicking the internal juxtamembrane domain of HER1 (EGFR; EJ1) promotes the formation of non-functional HER dimers that inhibit kinase-dependent and kinase-independent functions of HER1 (ERBB1/EGFR), HER2 (ERBB2) and HER3 (ERBB3). Despite inducing rapid HER-dependent cell death in vitro, EJ1 peptides are rapidly cleared in vivo, limiting their efficacy. METHOD: To stabilize EJ1 activity, hydrocarbon staples (SAH) were added to the active peptide (SAH-EJ1), resulting in a 7.2-fold increase in efficacy and decreased in vivo clearance. Viability assays were performed across HER1 and HER2 expressing cell lines, therapeutic-resistant breast cancer cells, clinically relevant HER1-mutated lung cancer cells, and patient-derived glioblastoma cells, in all cases demonstrating improved efficacy over standard of care pan-HER therapeutics. Tumor burden studies were also performed in lung, glioblastoma, and inflammatory breast cancer mouse models, evaluating tumor growth and overall survival. RESULTS: When injected into mouse models of basal-like and inflammatory breast cancers, EGFRvIII-driven glioblastoma, and lung adenocarcinoma with Erlotinib resistance, tumor growth is inhibited and overall survival is extended. Studies evaluating the toxicity of SAH-EJ1 also demonstrate a broad therapeutic window. CONCLUSIONS: Taken together, these data indicate that SAH-EJ1 may be an effective therapeutic for HER-driven cancers with the potential to eliminate triple negative inflammatory breast cancer.

Intravesicular epidermal growth factor receptor subject to retrograde trafficking drives epidermal growth factor-dependent migration.

The Epidermal Growth Factor Receptor (EGFR) is frequently mutated and overexpressed in metastatic cancer. Although EGFR is a transmembrane tyrosine kinase localized to the basolateral membrane in normal epithelium, it is frequently found intracellularly localized in transformed cells. We have previously demonstrated the epithelial adaptor protein mucin 1 (MUC1) alters trafficking of EGFR, inhibiting its degradation and promoting its translocation to the nucleus, where it can directly modulate gene transcription. Here, we demonstrate that MUC1 promotes the retention of EGF-bound EGFR in Early Endosome Antigen1 (EEA1)-positive vesicles while preventing its trafficking to the lysosome. These events result in the accumulation of endosomal vesicles harboring active receptor throughout the cell and a reorganization of the actin cytoskeleton. EGF-dependent cell migration and filopodia formation is reliant upon this altered trafficking, and can be prevented by blocking retrograde trafficking. Together, these results indicate that intracellular EGFR may play an essential role in cancer metastasis and a potential mechanism for the failure of therapeutic antibodies in EGFR-driven metastatic breast cancer.

Significance of Low-Attenuation Cluster Analysis on Quantitative CT in the Evaluation of Chronic Obstructive Pulmonary Disease.

Objective: To assess clinical feasibility of low-attenuation cluster analysis in evaluation of chronic obstructive pulmonary disease (COPD). Materials and Methods: Subjects were 199 current and former cigarette smokers that underwent CT for quantification of COPD and had physiological measurements. Quantitative CT (QCT) measurements included low-attenuation area percent (LAA%) (voxels ≤ -950 Hounsfield unit [HU]), and two-dimensional (2D) and three-dimensional D values of cluster analysis at three different thresholds of CT value (-856, -910, and -950 HU). Correlation coefficients between QCT measurements and physiological indices were calculated. Multivariable analyses for percentage of predicted forced expiratory volume at one second (%FEV1) was performed including sex, age, body mass index, LAA%, and D value had the highest correlation coefficient with %FEV1 as independent variables. These analyses were conducted in subjects including those with mild COPD (global initiative of chronic obstructive lung disease stage = 0-II). Results: LAA% had a higher correlation coefficient (-0.549, p < 0.001) with %FEV1 than D values in subjects while 2D D-910HU (-0.350, p < 0.001) revealed slightly higher correlation coefficient than LAA% (-0.343, p < 0.001) in subjects with mild COPD. Multivariable analyses revealed that LAA% and 2D D value-910HU were significant independent predictors of %FEV1 in subjects and that only 2D D value-910HU revealed a marginal p value (0.05) among independent variables in subjects with mild COPD. Conclusion: Low-attenuation cluster analysis provides incremental information regarding physiologic severity of COPD, independent of LAA%, especially with mild COPD.

miR-9a mediates the role of Lethal giant larvae as an epithelial growth inhibitor in Drosophila.

Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which we found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, we overexpressed it in the context of lgl knock-down by RNAi and found it able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. Our work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.

Idiopathic Pulmonary Fibrosis: Data-driven Textural Analysis of Extent of Fibrosis at Baseline and 15-Month Follow-up.

Purpose To evaluate associations between pulmonary function and both quantitative analysis and visual assessment of thin-section computed tomography (CT) images at baseline and at 15-month follow-up in subjects with idiopathic pulmonary fibrosis (IPF). Materials and Methods This retrospective analysis of preexisting anonymized data, collected prospectively between 2007 and 2013 in a HIPAA-compliant study, was exempt from additional institutional review board approval. The extent of lung fibrosis at baseline inspiratory chest CT in 280 subjects enrolled in the IPF Network was evaluated. Visual analysis was performed by using a semiquantitative scoring system. Computer-based quantitative analysis included CT histogram-based measurements and a data-driven textural analysis (DTA). Follow-up CT images in 72 of these subjects were also analyzed. Univariate comparisons were performed by using Spearman rank correlation. Multivariate and longitudinal analyses were performed by using a linear mixed model approach, in which models were compared by using asymptotic χ2 tests. Results At baseline, all CT-derived measures showed moderate significant correlation (P < .001) with pulmonary function. At follow-up CT, changes in DTA scores showed significant correlation with changes in both forced vital capacity percentage predicted (ρ = -0.41, P < .001) and diffusing capacity for carbon monoxide percentage predicted (ρ = -0.40, P < .001). Asymptotic χ2 tests showed that inclusion of DTA score significantly improved fit of both baseline and longitudinal linear mixed models in the prediction of pulmonary function (P < .001 for both). Conclusion When compared with semiquantitative visual assessment and CT histogram-based measurements, DTA score provides additional information that can be used to predict diminished function. Automatic quantification of lung fibrosis at CT yields an index of severity that correlates with visual assessment and functional change in subjects with IPF. © RSNA, 2017.

Quantitative computed tomography measurements to evaluate airway disease in chronic obstructive pulmonary disease: Relationship to physiological measurements, clinical index and visual assessment of airway disease.

PURPOSE: To correlate currently available quantitative CT measurements for airway disease with physiological indices and the body-mass index, airflow obstruction, dyspnea, and exercise capacity (BODE) index in patients with chronic obstructive pulmonary disease (COPD). MATERIALS AND METHODS: This study was approved by our institutional review board (IRB number 2778). Written informed consent was obtained from all subjects. The subjects included 188 current and former cigarette smokers from the COPDGene cohort who underwent inspiratory and expiratory CT and also had physiological measurements for the evaluation of airflow limitation, including FEF25-75%, airway resistance (Raw), and specific airway conductance (sGaw). The BODE index was used as the index of clinical symptoms. Quantitative CT measures included % low attenuation areas [% voxels≤950 Hounsfield unit (HU) on inspiratory CT, %LAA-950ins], percent gas trapping (% voxels≤-856HU on expiratory CT, %LAA -856exp), relative inspiratory to expiratory volume change of voxels with attenuation values from -856 to -950HU [Relative Volume Change (RVC)-856 to -950], expiratory to inspiratory ratio of mean lung density (E/I-ratio MLD), Pi10, and airway wall thickness (WT), luminal diameter (LD) and airway wall area percent (WA%) in the segmental, subsegmental and subsubsegmental bronchi on inspiratory CT. Correlation coefficients were calculated between the QCT measurements and physiological measurements in all subjects and in the subjects with mild emphysema (%LAA-950ins <10%). Univariate and multiple variable analysis for the BODE index were also performed. Adjustments were made for age, gender, smoking pack years, FEF25-75%, Raw, and sGaw. RESULTS: Quantitative CT measurements had significant correlations with physiological indices. Among them, E/I-ratio MLD had the strongest correlations with FEF25-75% (r=-0.648, <0.001) and sGaw (r=-0.624, <0.001) while in the subjects with mild emphysema subsegmental WA% and segmental WA% had the strongest correlation with FEF25-75% (r=-0.669, <0.001) and sGaw (r=-0.638, <0.001), respectively. The multiple variable analyses showed that RVC-856 to -950 was an independent predictor of the BODE index showing the highest R2 (0.468) as an independent variable among the QCT measurements. CONCLUSION: Quantitative CT measurements of gas trapping such as E/I-ratio MLD, correlate better with physiological indices for airway disease than those of airway such as WA% or LD. In mild emphysema, however, quantitative CT measurements of airway correlate better with the physiological indices. RVC-856 to -950 is a predictor of the BODE index.

Texture-based Quantification of Centrilobular Emphysema and Centrilobular Nodularity in Longitudinal CT Scans of Current and Former Smokers.

RATIONALE AND OBJECTIVES: The effect of smoking cessation on centrilobular emphysema (CLE) and centrilobular nodularity (CN), two manifestations of smoking-related lung injury on computed tomography (CT) images, has not been clarified. The objective of this study is to leverage texture analysis to investigate differences in extent of CLE and CN between current and former smokers. MATERIALS AND METHODS: Chest CT scans from 350 current smokers, 401 former smokers, and 25 control subjects were obtained from the multicenter COPDGene Study, a Health Insurance Portability and Accountability Act-compliant study approved by the institutional review board of each participating clinical study center. Additionally, for 215 of these subjects, a follow-up CT scan was obtained approximately 5 years later. For each CT scan, 5000 circular regions of interest (ROIs) of 35-pixel diameter were randomly selected throughout the lungs. The patterns present in each ROI were summarized by 50 computer-extracted texture features. A logistic regression classifier was leveraged to classify each ROI as normal lung, CLE, or CN, and differences in the percentages of normal lung, CLE, and CN by study group were assessed. RESULTS: Former smokers had significantly more CLE (P <0.01) but less CN (P <0.001) than did current smokers, even after adjustment for important covariates such as patient age, GOLD stage, smoking history, forced expiratory volume in 1 second, gas trapping, and scanner model. Among patients with longitudinal CT scans, continued smoking led to a slight increase in CLE (P = 0.13), whereas sustained abstinence from smoking led to further reduction in CN (P <0.05). CONCLUSIONS: The proposed texture-based approach quantifies the extent of CN and CLE with high precision. Differences in smoking-related lung disease between longitudinal scans of current smokers and longitudinal scans of former smokers suggest that CN may be reversible on smoking cessation.

Llgl1 prevents metaplastic survival driven by epidermal growth factor dependent migration.

We have previously demonstrated that Llgl1 loss results in a gain of mesenchymal phenotypes and a loss of apicobasal and planar polarity. We now demonstrate that these changes represent a fundamental shift in cellular phenotype. Llgl1 regulates the expression of multiple cell identity markers, including CD44, CD49f, and CD24, and the nuclear translocation of TAZ and Slug. Cells lacking Llgl1 form mammospheres, where survival and transplantability is dependent upon the Epidermal Growth Factor Receptor (EGFR). Additionally, Llgl1 loss allows cells to grow in soft-agar and maintain prolonged survival as orthotopic transplants in NOD-SCIDmice. Lineage tracing and wound healing experiments demonstrate that mammosphere survival is due to enhanced EGF-dependent migration. The loss of Llgl1 drives EGFR mislocalization and an EGFR mislocalization point mutation (P667A) drives these same phenotypes, including activation of AKT and TAZ nuclear translocation. Together, these data indicate that the loss of Llgl1 results in EGFR mislocalization, promoting pre-neoplastic changes.

Opposing Regulation of the EGF Receptor: A Molecular Switch Controlling Cytomegalovirus Latency and Replication.

Herpesviruses persist indefinitely in their host through complex and poorly defined interactions that mediate latent, chronic or productive states of infection. Human cytomegalovirus (CMV or HCMV), a ubiquitous ß-herpesvirus, coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication. UL135 promotes reactivation from latency and virus replication, in part, by overcoming replication-suppressive effects of UL138. The mechanism by which UL135 and UL138 oppose one another is not known. We identified viral and host proteins interacting with UL138 protein (pUL138) to begin to define the mechanisms by which pUL135 and pUL138 function. We show that pUL135 and pUL138 regulate the viral cycle by targeting that same receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR). EGFR is a major homeostatic regulator involved in cellular proliferation, differentiation, and survival, making it an ideal target for viral manipulation during infection. pUL135 promotes internalization and turnover of EGFR from the cell surface, whereas pUL138 preserves surface expression and activation of EGFR. We show that activated EGFR is sequestered within the infection-induced, juxtanuclear viral assembly compartment and is unresponsive to stress. Intriguingly, these findings suggest that CMV insulates active EGFR in the cell and that pUL135 and pUL138 function to fine-tune EGFR levels at the cell surface to allow the infected cell to respond to extracellular cues. Consistent with the role of pUL135 in promoting replication, inhibition of EGFR or the downstream phosphoinositide 3-kinase (PI3K) favors reactivation from latency and replication. We propose a model whereby pUL135 and pUL138 together with EGFR comprise a molecular switch that regulates states of latency and replication in HCMV infection by regulating EGFR trafficking to fine tune EGFR signaling.