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Amanda L Richer,1 Jacqueline M Friel,1 Vashti M Carson,2 Landon J Inge,1 Timothy G Whitsett2 1Norton Thoracic Institute, St Joseph’s Hospital and Medical Center, 2Cancer and Cell Biology Division, Translational Genomics Research Institute, Phoenix, AZ, USA Abstract: Lung cancer remains the leading cause of cancer-related mortality worldwide. The application of next-generation genomic technologies has offered a more comprehensive look at the mutational landscape across the different subtypes of non-small cell lung cancer (NSCLC). A number of recurrent mutations such as TP53, KRAS, and epidermal growth factor receptor (EGFR) have been identified in NSCLC. While targeted therapeutic successes have been demonstrated in the therapeutic targeting of EGFR and ALK, the majority of NSCLC tumors do not harbor these genomic events. This review looks at the current treatment paradigms for lung adenocarcinomas and squamous cell carcinomas, examining genomic aberrations that dictate therapy selection, as well as novel therapeutic strategies for tumors harboring mutations in KRAS, TP53, and LKB1 which, to date, have been considered “undruggable”. A more thorough understanding of the molecular alterations that govern NSCLC tumorigenesis, aided by next-generation sequencing, will lead to targeted therapeutic options expected to dramatically reduce the high mortality rate observed in lung cancer. Keywords: non-small cell lung cancer, precision medicine, epidermal growth factor receptor, Kirsten rat sarcoma viral oncogene homolog, serine/threonine kinase 11, tumor protein p53

Background: There is no clinically relevant serological marker for the early detection of oesophageal adenocarcinoma (EAC) and its precursor lesion, Barrett's oesophagus (BE)., Objective: To develop and test a blood-based assay for EAC and BE., Design: Oesophageal MicroRNAs of BaRRett, Adenocarcinoma and Dysplasia ( EMERALD ) was a large, international, multicentre biomarker cohort study involving 792 patient samples from 4 countries (NCT06381583) to develop and validate a circulating miRNA signature for the early detection of EAC and high-risk BE. Tissue-based miRNA sequencing and microarray datasets (n=134) were used to identify candidate miRNAs of diagnostic potential, followed by validation using 42 pairs of matched cancer and normal tissues. The usefulness of the candidate miRNAs was initially assessed using 108 sera (44 EAC, 34 EAC precursors and 30 non-disease controls). We finally trained a machine learning model (XGBoost+AdaBoost) on RT-qPCR results from circulating miRNAs from a training cohort (n=160) and independently tested it in an external cohort (n=295)., Results: After a strict process of biomarker discovery and selection, we identified six miRNAs that were overexpressed in all sera of patients compared with non-disease controls from three independent cohorts of different nationalities (miR-106b, miR-146a, miR-15a, miR-18a, miR-21 and miR-93). We established a six-miRNA diagnostic signature using the training cohort (area under the receiver operating characteristic curve (AUROC): 97.6%) and tested it in an independent cohort (AUROC: 91.9%). This assay could also identify patients with BE among patients with gastro-oesophageal reflux disease (AUROC: 94.8%, sensitivity: 92.8%, specificity: 85.1%)., Conclusion: Using a comprehensive approach integrating unbiased genome-wide biomarker discovery and several independent experimental validations, we have developed and validated a novel blood test that might complement screening options for BE/EAC., Trial Registration Number: NCT06381583., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Context.—: Folate receptor-α (FRα, encoded by the FOLR1 gene) is overexpressed in several solid tumor types, including epithelial ovarian cancer (EOC), making it an attractive biomarker and target for FRα-based therapy in ovarian cancer., Objective.—: To describe the development, analytic verification, and clinical performance of the VENTANA FOLR1 Assay (Ventana Medical Systems Inc) in EOC., Design.—: We used industry standard studies to establish the analytic verification of the VENTANA FOLR1 Assay. Furthermore, the VENTANA FOLR1 Assay was used in the ImmunoGen Inc-sponsored SORAYA study to select patients for treatment with mirvetuximab soravtansine (MIRV) in platinum-resistant EOC., Results.—: The VENTANA FOLR1 Assay is highly reproducible, demonstrated by a greater than 98% overall percent agreement (OPA) for repeatability and intermediate precision studies, greater than 93% OPA for interreader and greater than 96% for intrareader studies, and greater than 90% OPA across all observations in the interlaboratory reproducibility study. The performance of the VENTANA FOLR1 Assay in the SORAYA study was evaluated by the overall staining acceptability rate, which was calculated using the number of patient specimens that were tested with the VENTANA FOLR1 Assay that had an evaluable result. In the SORAYA trial, data in patients who received MIRV demonstrated clinically meaningful efficacy, and the overall staining acceptability rate of the assay was 98.4%, demonstrating that the VENTANA FOLR1 Assay is safe and effective for selecting patients who may benefit from MIRV. Together, these data showed that the assay is highly reliable, consistently producing evaluable results in the clinical setting., Conclusions.—: The VENTANA FOLR1 Assay is a robust and reproducible assay for detecting FRα expression and identifying a patient population that derived clinically meaningful benefit from MIRV in the SORAYA study., (© 2024 College of American Pathologists.)

In DESTINY-Breast04 (DB-04), safety and efficacy of HER2-targeted antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd) in previously treated HER2-low unresectable/metastatic breast cancer were established. This manuscript describes the analytical validation of PATHWAY Anti-HER2/neu (4B5) Rabbit Monoclonal Primary Antibody (PATHWAY HER2 (4B5)) to assess HER2-low status and its clinical performance in DB-04. Preanalytical processing and tissue staining parameters were evaluated to determine their impact on HER2 scoring. The recommended antibody staining procedure provided the optimal tumor staining, and deviations in cell conditioning and/or antibody incubation times resulted in unacceptable negative control staining and/or HER2-low status changes. Comparisons between antibody lots, kit lots, instruments, and day-to-day runs showed overall percent agreements (OPAs) exceeding 97.9%. Inter-laboratory reproducibility showed OPAs of ≥97.4% for all study endpoints. PATHWAY HER2 (4B5) was utilized in DB-04 for patient selection using 1340 tumor samples (59.0% metastatic, 40.7% primary, (0.3% missing data); 74.3% biopsy, 25.7% resection/excisions). Overall, 77.6% (823/1060) of samples were HER2-low by both central and local testing, with the level of concordance differing by sample region of origin and collection date. In DB-04, the efficacy of T-DXd over chemotherapy of physician's choice was consistent, regardless of the characteristics of the sample used (primary or metastatic, archival, or newly collected, biopsy or excision/resection). These results demonstrate that PATHWAY HER2 (4B5) is precise and reproducible for scoring HER2-low status and can be used with multiple breast cancer sample types for reliably identifying patients whose tumors have HER2-low expression and are likely to derive clinical benefit from T-DXd., (© 2023. The Author(s).)

Introduction: Biomarker testing is increasingly crucial for patients with early-stage non-small cell lung cancer (eNSCLC). We explored biomarker test utilization and subsequent treatment in eNSCLC patients in the real-world setting., Methods: Using COTA's oncology database, this retrospective observational study included adult patients ≥ 18 years old diagnosed with eNSCLC (disease stage 0-IIIA) between January 1, 2011 and December 31, 2021. Date of first eNSCLC diagnosis was the study index date. We reported testing rates by index year for patients who received any biomarker test within 6 months of eNSCLC diagnosis and by each molecular marker. We also evaluated treatments received among patients receiving the five most common biomarker tests., Results: Among the 1031 eNSCLC patients included in the analysis, 764 (74.1%) received ≥ 1 biomarker test within 6 months of eNSCLC diagnosis. Overall, epidermal growth factor receptor (EGFR; 64%), anaplastic lymphoma kinase (ALK; 60%), programmed death receptor ligand 1 (PD-L1; 48%), ROS proto-oncogene 1 (ROS1; 46%), B-Raf proto-oncogene (40%), mesenchymal epithelial transition factor receptor (35%), Kirsten rat sarcoma viral oncogene (29%), RET proto-oncogene (22%), human epidermal growth factor receptor 2 (21%), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (20%) were the 10 most frequently tested biomarkers. The proportion of patients undergoing biomarker testing rose from 55.3% in 2011 to 88.1% in 2021. The most common testing methods were Sanger sequencing for EGFR (244, 37%), FISH (fluorescence in situ hybridization) for ALK (464, 75%) and ROS1 (357, 76%), immunohistochemical assay for PD-L1 (450, 90%), and next-generation sequencing testing for other biomarkers. Almost all the 763 patients who received the five most common biomarker tests had a test before the initiation of a systemic treatment., Conclusion: This study suggests a high biomarker testing rate among patients with eNSCLC in the US, with testing rates for various biomarkers increasing over the past decade, indicating a continuous trend towards the personalization of treatment decisions., (© 2023. The Author(s).)

Background: Identification of HER2 protein overexpression and/or amplification of the HER2 gene are required to qualify breast cancer patients for HER2 targeted therapies. In situ hybridization (ISH) assays that identify HER2 gene amplification function as a stand-alone test for determination of HER2 status and rely on the manual quantification of the number of HER2 genes and copies of chromosome 17 to determine HER2 amplification., Methods: To assist pathologists, we have developed the uPath HER2 Dual ISH Image Analysis for Breast (uPath HER2 DISH IA) algorithm, as an adjunctive aid in the determination of HER2 gene status in breast cancer specimens. The objective of this study was to compare uPath HER2 DISH image analysis vs manual read scoring of VENTANA HER2 DISH-stained breast carcinoma specimens with ground truth (GT) gene status as the reference. Three reader pathologists reviewed 220, formalin-fixed, paraffin-embedded (FFPE) breast cancer cases by both manual and uPath HER2 DISH IA methods. Scoring results from manual read (MR) and computer-assisted scores (image analysis, IA) were compared against the GT gene status generated by consensus of a panel of pathologists. The differences in agreement rates of HER2 gene status between manual, computer-assisted, and GT gene status were determined., Results: The positive percent agreement (PPA) and negative percent agreement (NPA) rates for image analysis (IA) vs GT were 97.2% (95% confidence interval [CI]: 95.0, 99.3) and 94.3% (95% CI: 90.8, 97.3) respectively. Comparison of agreement rates showed that the lower bounds of the 95% CIs for the difference of PPA and NPA for IA vs MR were -0.9% and -6.2%, respectively. Further, inter- and intra-reader agreement rates in the IA method were observed with point estimates of at least 96.7%., Conclusions: Overall, our data show that the uPath HER2 DISH IA is non-inferior to manual scoring and supports its use as an aid for pathologists in routine diagnosis of breast cancer., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Lung cancer is the deadliest cancer worldwide, mainly due to its advanced stage at the time of diagnosis. A non-invasive method for its early detection remains mandatory to improve patients' survival. Plasma levels of 351 proteins were quantified by Liquid Chromatography-Parallel Reaction Monitoring (LC-PRM)-based mass spectrometry in 128 lung cancer patients and 93 healthy donors. Bootstrap sampling and least absolute shrinkage and selection operator (LASSO) penalization were used to find the best protein combination for outcome prediction. The PanelomiX platform was used to select the optimal biomarker thresholds. The panel was validated in 48 patients and 49 healthy volunteers. A 6-protein panel clearly distinguished lung cancer from healthy individuals. The panel displayed excellent performance: area under the receiver operating characteristic curve (AUC) = 0.999, positive predictive value (PPV) = 0.992, negative predictive value (NPV) = 0.989, specificity = 0.989 and sensitivity = 0.992. The panel detected lung cancer independently of the disease stage. The 6-protein panel and other sub-combinations displayed excellent results in the validation dataset. In conclusion, we identified a blood-based 6-protein panel as a diagnostic tool in lung cancer. Used as a routine test for high- and average-risk individuals, it may complement currently adopted techniques in lung cancer screening.

Immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) have rapidly become integral to standard-of-care therapy for non-small cell lung cancer and other cancers. Immunohistochemical (IHC) staining of PD-L1 is currently the accepted and approved diagnostic assay for selecting patients for PD-L1/PD-1 axis therapies in certain indications. However, the inherent biological complexity of PD-L1 and the availability of several PD-L1 assays - each with different detection systems, platforms, scoring algorithms and cut-offs - have created challenges to ensure reliable and reproducible results based on subjective visual assessment by pathologists. The increasing adoption of computer technologies into the daily workflow of pathology provides an opportunity to leverage these tools towards improving the clinical value of PD-L1 IHC assays. This review describes several image analysis software programs of computer-aided PD-L1 scoring in the hope of driving further discussion and technological advancement in digital pathology and artificial intelligence approaches, particularly as precision medicine evolves to encompass accurate simultaneous assessment of multiple features of cancer cells and their interactions with the tumor microenvironment., (© 2020 The Authors.)

Glioblastoma multiforme (GBM) is the most common brain malignancies in adults. Most GBM patients succumb to the disease less than 1 year after diagnosis due to the highly invasive nature of the tumor, which prevents complete surgical resection and gives rise to tumor recurrence. The invasive phenotype also confers radioresistant and chemoresistant properties to the tumor cells; therefore, there is a critical need to develop new therapeutics that target drivers of GBM invasion. Amplification of EGFR is observed in over 50% of GBM tumors, of which half concurrently overexpress the variant EGFRvIII, and expression of both receptors confers a worse prognosis. EGFR and EGFRvIII cooperate to promote tumor progression and invasion, in part, through activation of the Stat signaling pathway. Here, it is reported that EGFRvIII activates Stat5 and GBM invasion by inducing the expression of a previously established mediator of glioma cell invasion and survival: fibroblast growth factor-inducible 14 (Fn14). EGFRvIII-mediated induction of Fn14 expression is Stat5 dependent and requires activation of Src, whereas EGFR regulation of Fn14 is dependent upon Src-MEK/ERK-Stat3 activation. Notably, treatment of EGFRvIII-expressing GBM cells with the FDA-approved Stat5 inhibitor pimozide blocked Stat5 phosphorylation, Fn14 expression, and cell migration and survival. Because EGFR inhibitors display limited therapeutic efficacy in GBM patients, the EGFRvIII-Stat5-Fn14 signaling pathway represents a node of vulnerability in the invasive GBM cell populations. Implications: Targeting critical effectors in the EGFRvIII-Stat5-Fn14 pathway may limit GBM tumor dispersion, mitigate therapeutic resistance, and increase survival. Mol Cancer Res; 16(7); 1185-95. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: The incidence of esophageal adenocarcinoma (EAC) has increased over the last several decades. Apart from mutations in TP53 gene, there are little data on genetic drivers of EAC. Liver kinase B1 (LKB1) has emerged as a multifunctional tumor suppressor regulating cell growth, differentiation, and metabolism. Somatic inactivation of LKB1 has been described in several tumor types; however, whether LKB1 inactivation has a role in EAC is unknown. Here we analyzed patient tumors to assess the prevalence of LKB1 loss in EAC., Methods: Chromosomal deletion and expression of LKB1 in EAC were investigated using publicly available genomic data. Protein expression was assessed by immunohistochemistry (IHC) analysis for LKB1 in a tissue microarray (TMA) containing esophageal tumor specimens, including EAC. LKB1 was suppressed in EAC cells to determine the effects on cell growth in vitro., Results: Analysis of EAC data in The Cancer Genome Atlas dataset revealed significant deletion of chromosome 19p13.3, containing the LKB1 gene locus. Single copy loss (shallow deletion) of LKB1 was present in 58% of EAC samples. Expression of LKB1 was significantly lower in EAC tumors compared with normal esophagus. IHC analysis showed reduced LKB1 protein expression in EAC. Suppression of LKB1 was sufficient to enhance EAC cell growth in vitro., Conclusions: Our data suggest that inactivation of LKB1 frequently occurs in EAC. Based on the reported oncogenic effects of LKB1 inactivation, our data indicate that LKB1 loss may play a significant role in EAC tumorigenesis, and point to the need for future studies., (Copyright © 2017 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

G 1 -S checkpoint loss contributes to carcinogenesis and increases reliance upon the G 2 -M checkpoint for adaptation to stress and DNA repair, making G 2 -M checkpoint inhibition a target for novel therapeutic development. AZD1775, an inhibitor against the critical G 2 -M checkpoint protein WEE1, is currently in clinical trials across a number of tumor types. AZD1775 and DNA-damaging agents have displayed favorable activity in several preclinical tumor models, often in the molecular context of TP53 loss. Whether AZD1775 efficacy is modulated by other molecular contexts remains poorly understood. The tumor suppressor serine/threonine kinase 11 ( LKB1/STK11 ) is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC) and is commonly comutated with oncogenic KRAS mutations. We investigated the preclinical effects of AZD1775 in the context of KRAS / LKB1 in NSCLC. Using NSCLC cell lines, we found that AZD1775 alone and in combination with DNA-damaging agents (e.g., cisplatin and radiation) decreased tumor cell viability in LKB1 -deficient NSCLC cells. In vitro, LKB1 deficiency enhanced DNA damage and apoptosis in response to AZD1775 exposure compared with wild-type LKB1 cells. In a genetically engineered mouse model of mutant Kras with concomitant loss of Lkb1 , combined AZD1775 and cisplatin extended overall survival compared with cisplatin alone. Our data suggest that lack of phosphorylation of LKB1 by ATM was involved in AZD1775-mediated cytotoxicity. Collectively, these findings provide a clinical application for AZD1775 with DNA-damaging agents in KRAS / LKB1 NSCLC. Cancer Res; 77(17); 4663-72. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The survival of patients diagnosed with glioblastoma (GBM), the most deadly form of brain cancer, is compromised by the proclivity for local invasion into the surrounding normal brain, which prevents complete surgical resection and contributes to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) superfamily, can stimulate glioma cell invasion and survival via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the transcription factor NF-κB. To discover small molecule inhibitors that disrupt the TWEAK-Fn14 signaling axis, we utilized a cell-based drug-screening assay using HEK293 cells engineered to express both Fn14 and a NF-κB-driven firefly luciferase reporter protein. Focusing on the LOPAC1280 library of 1280 pharmacologically active compounds, we identified aurintricarboxylic acid (ATA) as an agent that suppressed TWEAK-Fn14-NF-κB dependent signaling, but not TNFα-TNFR-NF-κB driven signaling. We demonstrated that ATA repressed TWEAK-induced glioma cell chemotactic migration and invasion via inhibition of Rac1 activation but had no effect on cell viability or Fn14 expression. In addition, ATA treatment enhanced glioma cell sensitivity to both the chemotherapeutic agent temozolomide (TMZ) and radiation-induced cell death. In summary, this work reports a repurposed use of a small molecule inhibitor that targets the TWEAK-Fn14 signaling axis, which could potentially be developed as a new therapeutic agent for treatment of GBM patients.

In epithelial cancers, carcinoma cells coexist with normal cells. Although it is known that the tumor microenvironment (TME) plays a pivotal role in cancer progression, it is not completely understood how the tumor influences adjacent normal epithelial cells. In this study, a three-dimensional co-culture system comprising non-transformed epithelial cells (MDCK) and transformed carcinoma cells (MSV-MDCK) was used to demonstrate that carcinoma cells sequentially induce preneoplastic lumen filling and epithelial-mesenchymal transition (EMT) in epithelial cysts. MMP-9 secreted by carcinoma cells cleaves cellular E-cadherin (encoded by CDH1) from epithelial cells to generate soluble E-cadherin (sE-cad), a pro-oncogenic protein. We show that sE-cad induces EGFR activation, resulting in lumen filling in MDCK cysts. Long-term sE-cad treatment induced EMT. sE-cad caused lumen filling by induction of the ERK signaling pathway and triggered EMT through the sustained activation of the AKT pathway. Although it is known that sE-cad induces MMP-9 release and consequent EGFR activation in tumor cells, our results, for the first time, demonstrate that carcinoma cells can induce sE-cad shedding in adjacent epithelial cells, which leads to EGFR activation and the eventual transdifferentiation of the normal epithelial cells., (© 2015. Published by The Company of Biologists Ltd.)

Objective: Obesity has been linked to esophageal adenocarcinoma (EAC). We hypothesize that adipokines, which are altered by obesity, could affect EAC growth rates and potentially serve as biomarkers of disease and targets for treatment. We have developed a potential murine model to investigate the effects of obesity-altered adipokines on EAC in vivo., Methods: Severe combined immune-deficient mice were fed either a high-fat diet (HFD) containing 60% animal fat, or a control diet with 10% animal fat, and monitored for weight gain for 5 weeks. All mice were subcutaneously implanted with EAC cells (OE33), and tumor volume was monitored for an additional 4 weeks by direct measurement and uptake of fluorescently labeled 2-D-deoxyglucose. At sacrifice, serum triglyceride levels and abdominal fat-pad weight were measured to assess obesity state. Adipokine levels were measured within abdominal fat of tumor-bearing mice., Results: Mice fed the HFD displayed increased body weight, visceral fat, and serum leptin and triglycerides. All mice developed tumors; OE33 EAC cells in HFD mice displayed increased growth rates, proliferation, and metabolic activity relative to tumors of EAC in control diet mice. Adipokine expression in the abdominal fat revealed distinct changes associated with the HFD and increased body weight., Conclusions: Ad libitum feeding of the HFD was correlated with more-proliferative EAC tumors in vivo. This phenotype was associated with alterations to secreted adipokines, representing a potential mechanism for our observations. Further studies are necessary to explore findings, as they have potential to improve treatment of EAC., (Copyright © 2015 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Five-year survival rates for non-small cell lung cancer (NSCLC) have seen minimal improvement despite aggressive therapy with standard chemotherapeutic agents, indicating a need for new treatment approaches. Studies show inactivating mutations in the LKB1 tumor suppressor are common in NSCLC. Genetic and mechanistic analysis has defined LKB1-deficient NSCLC tumors as a phenotypically distinct subpopulation of NSCLC with potential avenues for therapeutic gain. In expanding on previous work indicating hypersensitivity of LKB1-deficient NSCLC cells to 2-deoxy-D-glucose (2DG), we find that 2DG has in vivo efficacy in LKB1-deficient NSCLC using transgenic murine models of NSCLC. Deciphering of the molecular mechanisms behind this phenotype reveals that loss of LKB1 in NSCLC cells imparts increased sensitivity to pharmacological compounds that aggravate ER stress. In comparison to NSCLC cells with functional LKB1, treatment of NSCLC cells lacking LKB1 with the ER stress activators (ERSA), tunicamycin, brefeldin A or 2DG, resulted in aggravation of ER stress, increased cytotoxicity, and evidence of ER stress-mediated cell death. Based upon these findings, we suggest that ERSAs represent a potential treatment avenue for NSCLC patients whose tumors are deficient in LKB1., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The five-year survival rate in advanced non-small cell lung cancer (NSCLC) remains below ten percent. The invasive and metastatic nature of NSCLC tumor cells contributes to the high mortality rate, and as such the mechanisms that govern NSCLC metastasis is an active area of investigation. Two surface receptors that influence NSCLC invasion and metastasis are the hepatocyte growth factor receptor (HGFR/MET) and fibroblast growth factor-inducible 14 (FN14). MET protein is over-expressed in NSCLC tumors and associated with poor clinical outcome and metastasis. FN14 protein is also elevated in NSCLC tumors and positively correlates with tumor cell migration and invasion. In this report, we show that MET and FN14 protein expressions are significantly correlated in human primary NSCLC tumors, and the protein levels of MET and FN14 are elevated in metastatic lesions relative to patient-matched primary tumors. In vitro, HGF/MET activation significantly enhances FN14 mRNA and protein expression. Importantly, depletion of FN14 is sufficient to inhibit MET-driven NSCLC tumor cell migration and invasion in vitro. This work suggests that MET and FN14 protein expressions are associated with the invasive and metastatic potential of NSCLC. Receptor-targeted therapeutics for both MET and FN14 are in clinical development, the use of which may mitigate the metastatic potential of NSCLC.

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett's esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

Ionophores are hydrophobic organic molecules that disrupt cellular transmembrane potential by permeabilizing membranes to specific ions. Gramicidin A is a channel-forming ionophore that forms a hydrophilic membrane pore that permits the rapid passage of monovalent cations. Previously, we found that gramicidin A induces cellular energy stress and cell death in renal cell carcinoma (RCC) cell lines. RCC is a therapy-resistant cancer that is characterized by constitutive activation of the transcription factor hypoxia-inducible factor (HIF). Here, we demonstrate that gramicidin A inhibits HIF in RCC cells. We found that gramicidin A destabilized HIF-1α and HIF-2α proteins in both normoxic and hypoxic conditions, which in turn diminished HIF transcriptional activity and the expression of various hypoxia-response genes. Mechanistic examination revealed that gramicidin A accelerates O(2)-dependent downregulation of HIF by upregulating the expression of the von Hippel-Lindau (VHL) tumor suppressor protein, which targets hydroxylated HIF for proteasomal degradation. Furthermore, gramicidin A reduced the growth of human RCC xenograft tumors without causing significant toxicity in mice. Gramicidin A-treated tumors also displayed physiologic and molecular features consistent with the inhibition of HIF-dependent angiogenesis. Taken together, these results demonstrate a new role for gramicidin A as a potent inhibitor of HIF that reduces tumor growth and angiogenesis in VHL-expressing RCC.

The high mortality rate in advanced lung cancer, due to a preponderance of tumors discovered at advanced stage, demands the discovery and clinical validation of biomarkers for diagnosing early stage disease. Quantitative proteomics technologies are capable of identifying protein biomarkers with diagnostic, prognostic, and predictive value. Recent works have demonstrated the utility in using quantitative proteomics across normal, pre-cancerous, and cancerous lesions towards the discovery of biomarkers for early stage lung cancer, as well as discovering novel mechanisms of lung carcinogenesis.

Lung cancer remains the leading cause of cancer-related mortality worldwide. The propensity for metastasis to the central nervous system (CNS) is a major clinical hurdle contributing to the low five-year survival rate of advanced disease. CNS metastases significantly outnumber primary brain tumors and carry a dismal prognosis in part due to the inability of therapeutic agents to cross the blood brain barrier. Standard treatment using radiation has been largely ineffective in improving mortality, suggesting the need for new agents targeting the critical metastatic drivers. The genetic and molecular events governing CNS metastasis from the lung are poorly understood at this time. This review highlights genetic events associated with CNS dissemination from the lung and molecular mechanisms associated with CNS metastasis. In vivo model systems that faithfully recapitulate escape from the lung and colonization of the CNS are described as tools for understanding the metastatic phenotype and for testing new therapeutic agents. A deeper understanding of the mechanisms of lung cancer metastasis to the CNS is needed to elucidate novel therapeutic avenues towards the improvement of the mortality associated with advanced stage lung cancer.

Background: Only local ablation (radiofrequency ablation, cryotherapy) or esophagectomy currently is available to treat high-grade dysplasia in Barrett's esophagus. Alternative treatments, specifically chemopreventive strategies, are lacking. Our understanding of the molecular changes of high-grade dysplasia in Barrett's esophagus offers an opportunity to inhibit neoplastic progression of high-grade dysplasia in Barrett's esophagus. Increased activity of the Src kinase and deregulation of the tumor suppressor p27 are features of malignant cells and high-grade dysplasia in Barrett's esophagus. Src phosphorylates p27, inhibiting its regulatory function and increasing cell growth and proliferation. We hypothesized that a small molecule inhibitor of Src might reduce the growth and reverse Src-mediated deregulation of p27 in Barrett's esophagus cells., Methods: Immortalized Barrett's esophagus cell lines established from patient biopsies were treated with the Src kinase inhibitor dasatinib and evaluated for p27 localization and protein levels, as well as for effects on the cell cycle and apoptosis using flow cytometry, viability assays, and protein and RNA markers., Results: Dasatinib reduced both Src activation and p27 phosphorylation and increased p27 protein levels and nuclear localization. These effects correlated with decreased proliferation, cell-cycle arrest, and activation of apoptosis. Analysis of biopsies of patients with Barrett's esophagus revealed the presence of phosphorylated p27 in high-grade dysplasia, consistent with in vitro findings., Conclusions: Dasatinib has considerable antineoplastic effects on Barrett's esophagus cell lines carrying genetic markers associated with dysplasia, which correlates with the reversal of p27 deregulation. These findings suggest that dasatinib has potential as a treatment for patients with high-grade dysplasia and Barrett's esophagus and that p27 holds promise as a biomarker in the clinical use of dasatinib in patients with high-grade dysplasia and Barrett's esophagus., (Copyright © 2013 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Na,K-ATPase is a hetero-oligomer of an α- and a β-subunit. The α-subunit (Na,K-α) possesses the catalytic function, whereas the β-subunit (Na,K-β) has cell-cell adhesion function and is localized to the apical junctional complex in polarized epithelial cells. Earlier, we identified two distinct conserved motifs on the Na,K-β(1) transmembrane domain that mediate protein-protein interactions: a glycine zipper motif involved in the cis homo-oligomerization of Na,K-β(1) and a heptad repeat motif that is involved in the hetero-oligomeric interaction with Na,K-α(1). We now provide evidence that knockdown of Na,K-β(1) prevents lumen formation and induces activation of extracellular regulated kinases 1 and 2 (ERK1/2) mediated by phosphatidylinositol 3-kinase in MDCK cells grown in three-dimensional collagen cultures. These cells sustained cell proliferation in an ERK1/2-dependent manner and did not show contact inhibition at high cell densities, as revealed by parental MDCK cells. This phenotype could be rescued by wild-type Na,K-β(1) or heptad repeat motif mutant of Na,K-β(1), but not by the glycine zipper motif mutant that abrogates Na,K-β(1) cis homo-oligomerization. These studies suggest that Na,K-β(1) cis homo-oligomerization rather than hetero-oligomerization with Na,K-α(1) is involved in epithelial lumen formation. The relevance of these findings to pre-neoplastic lumen filling in epithelial cancer is discussed.

High levels of the soluble form of E-cadherin can be found in the serum of cancer patients and are associated with poor prognosis. Despite the possible predictive value of soluble E-cadherin, little is understood concerning its patho-physiological consequences in tumor progression. In this study, we show that soluble E-cadherin facilitates cell survival via functional interaction with cellular E-cadherin. Exposure of cells to a recombinant form of soluble E-cadherin, at a concentration found in cancer patient's serum, prevents apoptosis due to serum/growth factor withdrawal, and inhibits epithelial lumen formation, a process that requires apoptosis. Further, soluble E-cadherin-mediated cell survival involves activation of the epidermal growth factor receptor (EGFR) and EGFR-mediated activation of both phosphoinositide-3 kinase (PI3K)/AKT and ERK1/2 signaling pathways. These results are evidence of a complex functional interplay between EGFR and E-cadherin and also suggest that the presence of soluble E-cadherin in cancer patients' sera might have relevance to cell survival and tumor progression., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: Obliterative bronchiolitis is the predominant histopathologic finding in patients with chronic rejection after lung transplant. This fibroproliferative transformation within small airways of lung allograft is poorly understood; however, studies suggest epithelial-mesenchymal transition plays a role. Transplant immunosuppressive therapy has been shown to cause epithelial-mesenchymal transition in renal tubular epithelial cells, with subsequent fibrosis. This study explored whether immunosuppressive therapy contributes to epithelial-mesenchymal transition in airway epithelial cells., Methods: Bronchial epithelial cell line RL-65 was treated 3 to 5 days with several immunosuppressive agents, including cyclosporine (INN ciclosporin), tacrolimus, azathioprine, mycophenolic acid, sirolimus, prednisone, and transforming growth factor β1 as control. We then analyzed cells for presence of mesenchymal morphology and protein markers., Results: Treatment with cyclosporine, azathioprine, mycophenolate, and sirolimus resulted in elongated and irregular cell shape, and all but azathioprine showed loss of cell-cell adhesions relative to vehicle-treated cells. Expressions of extracellular matrix proteins, fibronectin and collagen, along with mesenchymal marker, vimentin, were significantly upregulated. Immunofluorescence showed loss of E-cadherin at cell membranes and cytoskeletal rearrangements typical of epithelial-mesenchymal transition. These immunosuppressive agents also increased transforming growth factor produced by cells; however, tacrolimus- and prednisone-treated cells maintained epithelial morphology, baseline levels of matrix protein expression, and transforming growth factor production levels., Conclusions: Overall, we found that certain immunosuppressive agents may contribute to partial epithelial-mesenchymal transition in bronchial epithelial cells, specifically increasing production of excessive extracellular matrix proteins. This may provide novel insights into the pathogenesis of obliterative bronchiolitis after lung transplant., (Copyright © 2011 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Epithelial-to-mesenchymal transition (EMT) is an important developmental process, participates in tissue repair, and occurs during pathologic processes of tumor invasiveness, metastasis, and tissue fibrosis. The molecular mechanisms leading to EMT are poorly understood. Although it is well documented that transforming growth factor (TGF)-beta plays a central role in the induction of EMT, the targets of TGF-beta signaling are poorly defined. We have shown earlier that Na,K-ATPase beta(1)-subunit levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. In this study, we provide evidence that Na,K-ATPase is a new target of TGF-beta(1)-mediated EMT in renal epithelial cells, a model system used in studies of both cancer progression and fibrosis. We show that following treatment with TGF-beta(1), the surface expression of the beta(1)-subunit of Na,K-ATPase is reduced, before well-characterized EMT markers, and is associated with the acquisition of a mesenchymal phenotype. RNAi-mediated knockdown confirmed the specific involvement of the Na,K-ATPase beta(1)-subunit in the loss of the epithelial phenotype and exogenous overexpression of the Na,K-ATPase beta(1)-subunit attenuated TGF-beta(1)-mediated EMT. We further show that both Na,K-ATPase alpha- and beta-subunit levels are highly reduced in renal fibrotic tissues. These findings reveal for the first time that Na,K-ATPase is a target of TGF-beta(1)-mediated EMT and is associated with the progression of EMT in cancer and fibrosis.

Objective: There are potentially deleterious sequelae of the physiologic response to surgical intervention. Some inflammatory cytokines can act as tumor growth factors, angiogenic and metastatic promoters, or both. Modulation of negative effects could improve outcomes from surgical intervention. The effects of surgical intervention on gene expression have not been fully elucidated. We assayed gene expression changes in an animal model of thoracotomy versus a sham operation and evaluated the ability of a cyclooxygenase inhibitor, celecoxib, to mediate these changes., Methods: Sixty adult male BALB/c mice were randomized into one of 3 experimental groups: sham operation with anesthesia only, thoracotomy incision, and thoracotomy incision with perioperative celecoxib administration. Six hours after surgical intervention, the animals were killed, and blood was collected. RNA pools from each group were labeled and hybridized to Mouse Whole Genome Microarrays. Gene expression profiles were analyzed to determine the effect of both surgical intervention and celecoxib treatment., Results: Surgical intervention initiated a robust gene expression response. We identified 867 transcripts that were found to be statistically significant (corrected P < .05) and differentially expressed at least 2-fold in response to surgical intervention. Celecoxib had a profound effect on this response, preserving close to baseline levels of expression for most of those genes., Conclusions: Surgical intervention has a dramatic effect on the expression of genes related to the inflammatory response. Perioperative treatment with a cyclooxygenase 2 inhibitor abated many of these changes and might counteract many of the negative effects of the response to surgical intervention., (2010 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Objective: Targeted therapy promises to improve patient outcome in non-small cell lung cancer. Biomarkers can direct targeted therapy toward patients who are most likely to respond, thus optimizing benefit. A novel agent with antineoplastic potential is the glucose analog, 2-deoxyglucose. 2-Deoxyglucose targets tumor cells, owing to their increased glucose uptake, inhibiting cellular metabolism and inducing energetic stress, resulting in decreased cellular viability. The tumor suppressor LKB1 is activated by energetic stress, and cells that lack LKB1 fail to respond and undergo cell death, suggesting that LKB1-null non-small cell lung cancer may have an increased susceptibility to 2-deoxyglucose. Inasmuch as somatic loss of LKB1 is a frequent event in non-small cell lung cancer, LKB1 expression could be used as a biomarker for directing 2-deoxyglucose therapy in patients with this type of cancer., Methods: LKB1-positive and LKB1-negative non-small cell lung cancer cell lines were evaluated for cell viability, markers of apoptosis, and gene expression after 2-deoxyglucose treatment and compared with vehicle control., Results: LKB1-negative cells treated with 2-deoxyglucose displayed a significant decrease in cell viability compared with LKB1-positive cells. Gene expression profiles of 2-deoxyglucose treated cells revealed changes in apoptotic markers in LKB1-negative cells, correlating with activation of apoptosis. Re-expression of LKB1 prevented 2-deoxyglucose mediated apoptosis, demonstrating the critical role of LKB1 in mediating 2-deoxyglucose toxicity., Conclusions: LKB1 loss increases susceptibility to 2-deoxyglucose treatment in non-small cell lung cancer lines, even at low doses. Thus, determination of LKB1 status may help direct therapy to those patients most likely to benefit from this novel approach, making it useful in the treatment of patients with non-small cell lung cancer.

Loss of alpha-catenin is one of the characteristics of prostate cancer. The catenins (alpha and beta) associated with E-cadherin play a critical role in the regulation of cell-cell adhesion. Tyrosine phosphorylation of beta-catenin dissociates it from E-cadherin and facilitates its entry into the nucleus, where beta-catenin acts as a transcriptional activator inducing genes involved in cell proliferation. Thus, beta-catenin regulates cell-cell adhesion and cell proliferation. Mechanisms controlling the balance between these functions of beta-catenin invariably are altered in cancer. Although a wealth of information is available about beta-catenin deregulation during oncogenesis, much less is known about how or whether alpha-catenin regulates beta-catenin functions. In this study, we show that alpha-catenin acts as a switch regulating the cell-cell adhesion and proliferation functions of beta-catenin. In alpha-catenin-null prostate cancer cells, reexpression of alpha-catenin increased cell-cell adhesion and decreased beta-catenin transcriptional activity, cyclin D1 levels, and cell proliferation. Further, Src-mediated tyrosine phosphorylation of beta-catenin is a major mechanism for decreased beta-catenin interaction with E-cadherin in alpha-catenin-null cells. alpha-Catenin attenuated the effect of Src phosphorylation by increasing beta-catenin association with E-cadherin. We also show that alpha-catenin increases the sensitivity of prostate cancer cells to a Src inhibitor in suppressing cell proliferation. This study reveals for the first time that alpha-catenin is a key regulator of beta-catenin transcriptional activity and that the status of alpha-catenin expression in tumor tissues might have prognostic value for Src targeted therapy.

The Na,K-ATPase, consisting of two essential subunits (alpha, beta), plays a critical role in the regulation of ion homeostasis in mammalian cells. Recent studies indicate that reduced expression of the beta1 isoform (NaK-beta1) is commonly observed in carcinoma and is associated with events involved in cancer progression. In this study, we present evidence that repletion of NaK-beta1 in Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK), a highly tumorigenic cell line, inhibits anchorage independent growth and suppresses tumor formation in immunocompromised mice. Additionally, using an in vitro cell-cell aggregation assay, we showed that cell aggregates of NaK-beta1 subunit expressing MSV-MDCK cells have reduced extracellular regulated kinase (ERK) 1/2 activity compared with parental MSV-MDCK cells. Finally, using immunohistochemistry and fully quantitative image analysis approaches, we showed that the levels of phosphorylated ERK 1/2 are inversely correlated to the NaK-beta1 levels in the tumors. These findings reveal for the first time that NaK-beta1 has a potential tumor-suppressor function in epithelial cells.

Studies of neurons grafted into the brains of experimental animals have been limited by the lack of suitably homogeneous populations of neurons for transplantation. Here we describe the transplantation and survival of pure, postmitotic human neurons (NT2N cells) into the rat brain. NT2N cells were derived from a human teratocarcinoma line (NTera2/clone D1 or NT2 cells) in vitro by retinoic acid treatment. Approximately 5-10 x 10(4) NT2N cells (including previously frozen aliquots of NT2N cells) were injected into the neocortex, subjacent white matter, or hippocampus of adult (N = 51) or neonatal (N = 17) Sprague-Dawley rats. Cyclosporine (7-10 mg/kg) was administered daily to 13 adult rats for up to 12 weeks post-transplant prior to sacrifice. Untreated rats survived for up to 21 weeks post-transplant. Injection sites were serially sectioned and NT2N grafts were analyzed immunohistochemically using antibodies to diverse neuronal and glial proteins to assess the lineage of the grafted cells and their ability to establish molecular and structural polarity. NT2N cells transplanted into untreated adult and neonatal rat brains were committed exclusively to the neuronal phenotype and survived for as long as 8 weeks, although most were rejected after 4 weeks. However, cyclosporine prolonged survival of the NT2N grafts for up to 12 weeks. Further, grafted NT2N cells exhibited an asymmetric geometry (with long axons and simplified dendrites), as well as molecular polarity (with highly phosphorylated neurofilament proteins segregated in axons and microtubule associated protein 2 confined to perikarya and dendrites) by 4 weeks post-transplant. However, the grafted neurons did not become fully mature as evidenced by their failure to express the most highly phosphorylated heavy neurofilament proteins. Finally, previously frozen NT2N cells survived in the rat brain, and none of the grafts formed neoplasms. We conclude from these studies that transplanted NT2N cells represent a highly advantageous model system for studies of the developmental biology of neurons.