Lung carcinoid cell lines have Bombesin‐like peptides and EGF receptors

e20101 Background: The RUNX1T1 gene, also known as ETO, was first identified in the fusion transcript AML1/ETO, generated by the translocation between chromosome 8 and 21, which occurs in 12%-15% of AML. We recently discovered that RUNX1T1 was specifically amplified only in the small cell lung cancer (SCLC) but not the non-small cell lung cancer (NSCLC) component of combined SCLC tumors. Here, we investigated if RUNX1T1 expression plays a role in transforming NSCLC to SCLC in combined SCLC tumor formation. Methods: We examined tumors from 92 of our SCLC patients by targeted exome and CNV analyses. RUNX1T1 mRNA expression was determined in various cancers by analysis of the CCLE and TCGA databases. RUNX1T1 mRNA expression was detected by RNAscope analysis of another 22 patient SCLC tumors. Stable overexpression of RUNX1T1 in NSCLC and SCLC cell lines used commercial RUNX1T1 lentiviral constructs. The resulting stable cell lines were analyzed by RT-qPCR, western blotting, Affymetrix gene array, and E2F-luciferase assays. Results: We found that 2 of 2 patients with combined SCLC demonstrated RUNX1T1 amplification in the SCLC component. Notably, only 2 of the remaining 90 patients with pure SCLC demonstrated RUNX1T1 amplification. RUNX1T1 mRNA is highly expressed in SCLC compared to the majority of other cancers, including NSCLC, using either CCLE or TCGA databases. Western blots confirmed higher RUNX1T1 protein expression in SCLC vs NSCLC cell lines, and RNAscope analysis confirmed greater RUNX1T1 mRNA expression in SCLC vs NSCLC patient tumors. When we stably overexpressed RUNX1T1 in NSCLC cell lines, there was no increase in neuroendocrine protein expression, however we did find an increased sensitivity to etoposide. Bioinformatic analysis of microarray data demonstrated significantly altered E2F pathway activity, and we could show a significant increase in E2F-luciferase activity after RUNX1T1 overexpression. Interestingly, this was associated with decreased p21 expression, which is a putative suppressor of E2F activity. Conclusions: RUNX1T1 expression is specifically upregulated in SCLC compared to NSCLC. E2F pathway activity is elevated after RUNX1T1 overexpression, consistent with an increased SCLC phenotype.

Purpose: The intent of the study described herein is to develop and characterize a microRNA gene expression signature of response to EGFR inhibition portable to solid tumors for which treatment with an EGFR inhibitor is indicated. Experimental Design: Treatment with inhibitors of the epidermal growth factor receptor (EGFR) can result in clinical response in both non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC), but only in a minority of unselected patients. Identification of biomarkers that can direct treatment to the responsive population is essential and here we describe generation of a microRNA gene signature of response to EGFR inhibition. Using a Taqman MicroRNA Array of 381 probes, we demonstrate that a 20-gene microRNA signature derived from NSCLC cell lines can predict sensitivity of both lung and pancreatic cancer cell lines to erlotinib, a small molecule inhibitor of EGFR. Overlap of independently generated microRNA expression signatures of NSCLC and pancreatic cell lines produces a fourgene signature. We characterize a target of the reduced signature, ZEB1, in NSCLC and pancreatic cancer cell lines by evaluation of mRNA and protein expression. We evaluated ZEB1 protein and the ZEB1 target, E-cadherin, after transfection of mir-200c into inhibitor-resistant NSCLC cell line, A549. Results: We have identified a 20-gene microRNA signature of response to EGFR inhibition that predicts both inhibitor-sensitive NSCLC and pancreatic cell lines. By overlapping independent signatures of response in NSCLC and pancreatic cancer cell lines, a four gene microRNA signature is identified that targets proteins responsible for epithelial to mesenchymal transition (EMT). Of those proteins, ZEB1 is a transcription factor responsible for repression of E-cadherin expression. Both ZEB1 mRNA and protein expression are down-regulated in erlotinib-sensitive NSCLC in which the microRNA genes 200b, 200c, and 141 are up-regulated. Ectopic expression of mir-200c in inhibitor-resistant lines induces cell death, suggesting that down-regulation of this microRNA is essential for viability in these NSCLC lines. Conclusions: The 20-gene miRNA signature of sensitivity provides a means for defining response that can be generated from FFPE samples of tumor rather than fresh tumor samples. Whereas mRNA is difficult to isolate from FFPE samples due to degradation, microRNA may survive the fixation process. Thus, response to EGFR inhibition in the second- or third-line can be predicted from fixed tumor samples collected early in the treatment of an individual patient, eliminating the need for acquisition of fresh tumor. Further, the microRNA signature identifies the well-studied EMT pathway indicative of EGFR-inhibitor sensitivity and provides biological significance for the members of the signature. Citation Information: Cancer Res 2009;69(23 Suppl):B74.

This study aims to investigate the impacts of SLC12A8 on the invasion, migration, and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) cells. GEPIA database was employed to examine SLC12A8 expression pattern in lung cancer cells. Subsequently, qRT-PCR and Western blot analyses were conducted to assess SLC12A8 expression in NSCLC tissues and cell lines. The overall prognosis of NSCLC patients was evaluated using Kaplan-Meier plot and univariate and multivariate COX regression curves. The knockdown of SLC12A8 was established using lentivirus-mediated shRNA in A549 and H1299 cells. Cell proliferation, invasion, migration, and apoptosis were evaluated using CCK-8 assay, transwell, and flow cytometry techniques, respectively. Western blot analysis was performed to measure the expression levels of EMT-related proteins (E-cadherin and vimentin). The expression level of SLC12A8 was found to be significantly higher in both NSCLC cell lines and tissues. High SLC12A8 expression was correlated with a poor prognosis in NSCLC patients. Knocking down SLC12A8 led to a significant decrease in proliferation, migration, and invasion abilities, while promoting apoptosis in NSCLC cells. Additionally, SLC12A8 knockdown resulted in decreased levels of N-cadherin and vimentin, along with increased E-cadherin expression. The results indicate that reducing SLC12A8 expression may suppress the malignant phenotype of NSCLC cells, as well as the EMT. SLC12A8 may serve as a target for the clinical management of NSCLC progression.

Human complement factor H (CFH), a central complement control protein, is a member of the regulators of complement activation family. Recent studies suggested that CFH may play a key role in the resistance of complement-mediated lysis in various cancer cells. In this study, we investigated the role of CFH in human lung cancer. Expression of CFH was analyzed in lung cancer cell lines by RT-PCR, Western blotting and immunofluorescence. In primary lung tumors, the protein expression of CFH was evaluated by immunohistochemistry (IHC) on tissue microarray (TMA). Binding of CFH to lung cancer cells was detected by flow cytometry. mRNA expression of CFH was detected in 6 out of 10 non-small cell lung cancer (NSCLC) cell lines, but in none of the small cell lung cancer (SCLC) cell lines. In line with Western blotting, immunofluorescence analysis demonstrated CFH protein expression in 3 NSCLC cell lines, and the immunoreaction was mainly associated with cell cytoplasm and membrane. In primary lung tumors, 54 out of 101 samples exhibited high expression of CFH and high expression was significantly correlated with lung adenocarcinoma (p=0.009). Also, in adenocarcinoma of the lung, Kaplan-Meier survival analysis showed a tendency that CFH-positive tumors had worse prognosis in comparison to CFH-negative tumors (p=0.082). Additionally, shorter survival time of patients with adenocarcinoma (<20 months) was associated with higher staining of CFH (p=0.033). Our data showed that non-small cell lung cancer cells expressed and secreted CFH. CFH might be a novel diagnostic marker for human lung adenocarcinoma.

Expression of Inhibitor of Apoptosis Proteins in Small- and Non-Small-Cell Lung Carcinoma Cells

Background: The cell cycle progression of which alteration is well known in human cances involves sequential activation and inactivation of cyclin-dependent kinases. The p21 (CDKN1A) plays a key role in regulating the cell cycle. Loss of expression or function of p21 (CDKN1A) has been implicated in the genesis or progression of many human malignancies. DNA methylation in tumor suppressor gene has been established as an important mechanism for gene silencing in human malignancies. In this study, we examined the methylation and expression status of P21 gene in lung cancer and malignant pleural mesothelioma (MPM). Methods: We used 14 non-small-cell lung cancer (NSCLC) cell lines and 20 primary tumors, 12 small-cell lung cancer (SCLC) cell lines, 7 MPM cell lines and 10 primary MPMs. Methylation of P21 gene was examined with methylation-specific PCR assay. p21 expression was examined by RT-PCR and western blotting. Cell lines with methylation were treated with 5-aza-CdR to confirm restoration of p21 expression. Results: Aberrant methylation of P21 was found in 20.6% of NSCLCs (28.6% and 15% in cell lines and primary NSCLCs, respectively), 8.3% of SCLC cell lines, 5.9% of MPMs (0% and 10% in MPM cell lines and primary MPMs, respectively). In NSCLC tumors, P21 methylation was detected only in smokers and squamous cell carcinoma cases but not in never smokers and adenocarinomas with bronchioloalveolar carcinoma feature. Loss of P21 mRNA expression were observed in 4 NSCLC cell lines (50.0%), 4 SCLC cell lines (33.3%) and none of MM cell lines (0%) while loss of protein expression was present in 10 NSCLC cell lines (83.3%), 6 SCLC cell lines (58.3%) and 2 MM cell lines (33.3%). mRNA expression of P21 was restored with 5-Aza-CdR treatment, indicating that methylation was responsible for silencing. These results suggested that 1) P21 methylation was one of the mechanisms of loss of P21 mRNA expression, and 2) post-transcriptional modification is responsible for loss of p21 protein expression. Conclusion: Aberrant methylation of p21 is present in lung cancer, especially for NSCLC, and rare in MPM. p21 protein expression was frequently loss in NSCLC and SCLC, partially due to DNA methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4938.

KRAS is the predominant form of mutated RAS (mut-KRAS) and is found in 25% of patient tumors across many cancer types. Mut-KRAS is well known to play a critical role in driving tumor growth and resistance to therapy, and its effects are so powerful that it overrides the activity of many of the new molecularly targeted signaling drugs being developed for cancer today such that they cannot be used in patients with mut-KRAS. However, despite extensive effort, there is no effective treatment for mut-KRAS. The effects of mut-KRAS are mediated through multiple downstream signaling pathways which have been independently associated with tumorigenesis, including RAF1, RALGDS, and PI3K. Current efforts to treat mut-KRAS tumors employ concurrent treatment with inhibitors of the RAF and PI3K pathways but this does not address the potential contribution of other pathways for which there are currently no inhibitors. Using a global siRNA screen we searched for genes that when inhibited would block the growth of mut-KRAS cancer cells without affecting wild type-KRAS (wt-KRAS) cell growth using an isogenic MiaPaCa-2 pancreatic cell line with and without oncogenic KRAS, and validated these hits in a similar isogenic HCT-116 colon cell line. From the screen we identified CNKSR1 (connector enhancer of kinase suppressor of Ras 1) as a top hit. CNKSR1 is found associated with KRAS in the RAS membrane associated signaling nanocluster that KRAS has to be associated with to provide growth signals. Knockdown of CNKSR1 with siRNA inhibited the growth of a panel of 10 mut-KRAS non small cell lung cancer (NSCLC) cell lines but not of 4 NSCLC cell lines with wt-KRAS. CNKSR1 is a multidomain protein that has a potentially druggable plekstrin homology (PH) domain responsible for binding to membrane phosphatidylinositols-3-phosphates. In order to demonstrate whether the PH domain of CNKSR1 is necessary for mut-KRAS activity we over expressed the PH domain in H1373 mut-KRAS NSCLC cells and found that it acted as a dominant negative and inhibited cell growth. We suggest that the PH domain fragment competes with the full length CNKSR1 in the cell. We also showed that knockdown of CNKSR1 inhibited KRAS dependent phosphorylation of RAF1 in multiple non small cell lung cancer cell lines. Together our results suggest that the CNKSR1 protein, acting through it's PH domain, is necessary for cell growth and down stream signaling by the KRAS oncogene. The PH domain can be targeted by inhibitors thus potentially providing agents that will selectively block mut-KRAS signaling and cell growth, creating a therapeutic potential for patients with oncogenic KRAS for which there is currently no effective therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A197.

Introduction: Lung cancer, although not the most commonly diagnosed form of cancer, is the leading cause of cancer deaths in the US. Unfortunately, standard chemotherapeutic treatments for lung cancer are largely ineffective and, thus, novel approaches are urgently needed to improve clinical outcomes. Targeted therapies are currently being explored, including combining bortezomib, a general proteasome inhibitor, with other therapies. The immunoproteasome is an alternative form of the proteasome and has been shown to play an important role in a number of diseases including cancer. We have previously developed ‘UK-101’, a small molecule that selectively inhibits the immunoproteasome catalytic subunit LMP2. In our present study, we investigate the therapeutic potential of the LMP2-targeting approach in lung cancer. Experimental Procedures: The expression and cellular localization of LMP2 was evaluated in primary non-small cell lung cancer (NSCLC) tissues via immunohistochemistry (IHC). Established lung cancer cell lines were likewise examined using western blotting (WB) and immunofluorescence (IF). A panel of NSCLC cell lines were selected for further analysis using UK-101 and the general proteasome inhibitors epoxomicin and bortezomib. Upon treatment with these inhibitors, cell viability was measured using the MTS assay while proteasome activity was interrogated using Suc-LLVY-aminoluciferin. Apoptosis markers were examined to determine the mechanism of UK-101-induced loss of cell viability. Finally, the presence of a naturally occurring LMP2 genetic polymorphism (Arg&amp;gt;His at codon 60) in the NSCLC cell lines was determined using restriction fragment length polymorphism analysis. Data Summary: Our WB and IHC analyses indicated that LMP2 is substantially upregulated in primary NSCLC tissues as well as established NSCLC cell lines compared to nonmalignant lung tissues and small cell lung cancer cell lines. IF and IHC analyses showed LMP2 localization throughout the cells, including overlap with nuclear staining in both. UK-101 treatment led to a covalent modification of LMP2 and decreased proteasomal activity. In addition, UK-101 decreased cell viability and induced apoptotic cell death in NSCLC cells expressing LMP2. The LMP2 polymorphism at codon 60 did not appear to influence the proteasomal activity or cellular sensitivity to UK-101, epoxomicin, or bortezomib in these NSCLC cell lines. Conclusion: Our results show that LMP2 is upregulated in NSCLC and that the targeted inhibition of LMP2 using UK-101 induces apoptotic cell death in NSCLC cell lines. Taken together, these results suggest that LMP2 may be a potential therapeutic target for the treatment of NSCLC with lesser systemic toxicity than general proteasome inhibitors. Further studies are warranted to identify molecular factors impacting the effectiveness of the immunoproteasome-targeting approaches in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2616. doi:10.1158/1538-7445.AM2011-2616

BackgroundThe plasticity of macrophage phenotype within the tumor microenvironment (TME) correlates with prognosis in non-small cell lung cancer (NSCLC).1 M2-like macrophages promote immunosuppression and facilitate tumor progression, while M1-like macrophages may drive an inflammatory antitumor immune response.2 Through a novel co-culture model comprised of cancer cells, cancer-associated fibroblasts (CAFs), and macrophages, we investigated whether NSCLC oncogenotype impacts macrophage phenotype and postulated that the immunosuppressive activity of macrophages is mediated through tumor-secreted soluble molecules. If identified and inhibited, these may re-sensitize cancer cells to immune surveillance and enhance antitumor immunity.MethodsWe developed an in vitro co-culture system (patient-derived NSCLC cells, human CAFs, and mouse macrophages) to interrogate impact of NSCLC cells and CAFs on macrophage phenotype. Expression of salient macrophage genes (i.e. ARG1, NOS2, IL-1β, IL-6, CHIL-3, SOCS3) was investigated through species-specific qPCR. Whole-genome RNA sequencing (RNAseq) in select cases was conducted and cytokine arrays measuring expression of 40 inflammatory cytokines were performed. Positive controls included stimulation of macrophages with LPS and IL-4.ResultsMore than 70 NSCLC cell lines were characterized in the co-culture assay. Three highly reproducible clusters of macrophage phenotypes were identified: high Arginase (immunosuppressive), high IL-1β (inflammatory) and high SOCS3 (inflammatory, involved in JAK-STAT3 pathway) (figure 1).3 4 Major oncogenotypes (i.e. KRAS, TP53, STK11, EGFR, BRAF mutation) did not correlate with macrophage phenotype (figure 2). Analyses of differences between the 3 clusters is ongoing. 10 exemplar NSCLC lines representing each of these 3 clusters were selected for RNA sequencing (mouse genes) and cytokine array protein (human) profiling. Across all clusters, we found suppression of macrophage endocytosis pathways and activation of scavenger receptor A (SRA) signaling, reflecting an M2-like phenotype.5 We also observed increased expression of human IL-6, IL-8, and MCP1, which are implicated in suppression of innate immune sensing of tumor cells (figure 3). RNAseq of CAF lines demonstrated mixed inflammatory and myofibroblastic phenotypes (figure 4), with increased expression of genes associated with macrophage recruitment and activation including: IL-6, CSF-1, CXCL6, CCL2, and CCL7.6Abstract 746 Figure 1Three macrophage phenotypes induced in co-cultureHeatmap of mRNA expression from mouse macrophages co-cultured with human NSCLC cells and CAFs. mRNA expression of salient mouse macrophage genes depicted (x-axis) for each NSCLC cell line co-culture (y-axis).Abstract 746 Figure 2Macrophage phenotype independent of oncogenotypePercentage of mutations of known human NSCLC oncogenes per mouse macrophage phenotype cluster.Abstract 746 Figure 3Upregulation of macrophage-related cytokinesCytokine array assays demonstrating relative expression of cytokines and chemokines from individual cell types or multicellular co-cultures associated with macrophage recruitment and polarizationAbstract 746 Figure 4Mixed expression of iCAF and myCAF genes on RNAseqHeatmap of RNAseq transcriptome of human CAFs from co-culture model reflecting relative expression of known genes associated inflammatory (iCAF, top) and myofibroblastic (myCAF, bottom) phenotypes.Abstract 746 Figure 5Novel co-culture model of NSCLC TMEDepiction of novel co-culture model with mouse bone-marrow derived macrophages, human NSCLC cells, and human CAFs with a representative immunohistochemical fluorescence image in vitroConclusionsThrough this novel co-culture model (figure 5), we demonstrate that patient-derived NSCLC cells reproducibly induce three major macrophage phenotypes in an oncotype-independent manner. Furthermore, cytokine release from NSCLC cells and CAFs is implicated in this process. This co-culture model provides a physiologically consistent experimental platform to identify tumor cell and CAF features that drive macrophage phenotype which may be suitable for targeted therapy.AcknowledgementsWe thank the McDermott Center Next-Generation Sequencing Core at UT Southwestern. Figure 5 was created with Biorender.comReferencesSumitomo R, Hirai T, Fujita M, et al. M2 tumor associated macrophages promote tumor progression in non small cell lung cancer. Exp Ther Med 2019 Dec 1;18(6):4490–8.Chen Y, Song Y, Du W, et al. Tumor-associated macrophages: an accomplice in solid tumor progression. J. Biomed. Sci 2019 Dec;26(1):1–3.Orecchioni M, Ghosheh Y, Pramod A, et al. Macrophage polarization: different gene signatures in M1 (LPS+) vs. classically and M2 (LPS–) vs. alternatively activated macrophages. Front. Immunol 2019 May 24;10:1084.Wilson HM. SOCS proteins in macrophage polarization and function. Front. Immunol 2014 Jul 28;5:357.Sun Y, Xu S. Tumor-associated CD204-positive macrophage is a prognostic marker in clinical stage I lung adenocarcinoma. Biomed Res. Int 2018 Jan 1;2018.O’Hayre M, Salanga C, Handel T, et al. Chemokines and cancer: migration, intracellular signalling and intercellular communication in the microenvironment. Biochem. J 2008 Feb 1;409(3):635–49

The protein kinase, membrane‑associated tyrosine/threonine 1 (PKMYT1) has been implicated as an important factor promoting the tumorigenesis of hepatocellular carcinoma and colorectal cancer. The current study was designed to explore the functional role of PKMYT1 in non-small cell lung cancer (NSCLC) cell behaviors and to investigate the possible molecular mechanisms. The expression levels of PKMYT1 in NSCLC tissues and cell lines were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The clinical and prognostic significance of PKMYT1 in 153 cases of NSCLC was determined. We also evaluated the effects of KMYT1 on NSCLC cell proliferation, migration, and invasion in vitro. Western blot was performed to assure whether PKMYT1 affected the Notch signal pathway and MET pathway. We observed that PKMYT1 expression was significantly up-regulated in both NSCLC tissues and cell lines. Higher expression of PKMYT1 was associated with clinical stage and lymph nodes metastasis. Clinical survival assays demonstrated that patients with high PKMYT1 expression had a shorter overall survival time than those with low PKMYT1 expression. Moreover, the multivariate analysis confirmed that increased expression of PKMYT1 was an independent predictor of overall survival. Functionally, knockdown of PKMYT1 in the NSCLC cell lines A549 and H1299 suppressed NSCLC cells proliferation, invasion and migration, and promoted apoptosis. In addition, the down-regulation of PKMYT1 resulted in the inhibition of EMT in NSCLC cells. Further mechanistic studies revealed that when PKMYT1 was silenced, the expression levels of Notch1, p21, and Hes1 were respectively downregulated, suggesting that PKMYT1 could promote the activity of the Notch signal pathway. PKMYT1 plays a significant role in NSCLC aggressiveness and clinical outcome, and may serve as a promising therapeutic target for this disease.

DNA methylation alteration of several genes contributes to human tumorigenesis. Cyclin D2, a member of the D-type cyclins, is implicated in cell cycle regulation and malignant transformation. In our study, we examined the methylation status of the cyclin D2 promoter in small cell lung cancer (SCLC), nonsmall cell lung cancer (NSCLC), breast tumors and tumor cell lines. We observed that aberrant methylation of cyclin D2 was present in 32 of 56 (57%) SCLC cell lines, 7 of 32 (22%) SCLC tumor tissues; 25 of 61 (47%) NSCLC cell lines, 19 of 48 (40%) NSCLC tumor tissues; 18 of 30 (60%) breast tumor cell lines and 19 of 63 (30%) breast tumor tissues. Methylation was more frequent in the tumor cell lines compared to the primary breast and SCLC tumors (p = 0.007 and p = 0.001, respectively). Methylation was rare in the control tissue samples; 0 of 12 peripheral blood lymphocytes; 0 of 12 buccal epithelial cells; 0 of 18 nonmalignant lung tissues and 3 of 28 (11%) nonmalignant breast tissues. Promoter methylation correlated with loss of transcript by reverse transcription PCR (RT-PCR) in 9 of 11 (6 lung, 5 breast) tumor cell lines tested. Two cell lines that were not methylated also lacked expression, suggesting that other mechanisms of inactivation may be involved. Expression was restored by treatment with the demethylating agent, 5 aza 2' deoxycytidine, in all 9 methylated cell lines. Our results confirm earlier reports in breast cancer and indicate that aberrant methylation of cyclin D2 may contribute to the pathogenesis of the 2 major types of lung cancers.

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130(Cas). Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130(Cas). WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.

Background: Antibody-IFNα fusion proteins represent a cancer therapeutic with properties of an antibody-drug conjugate and an immunotherapeutic agent, having both direct anti-tumor and immune-activating effects. These powerful molecules have the potential to target the anti-tumor cytotoxic effects of IFN to the tumor and to change the immunosuppressive tumor microenvironment to activate anti-tumor immunity. We previously reported the activity of IGN002, a recombinant anti-CD20-IFNα fusion protein, against human non-Hodgkin B-cell lymphomas (Yamada et al, JCO 2013). We now report the in vitro and in vivo anti-tumor activity of IGN004, an antibody-IFNα fusion protein against a novel tumor associated antigen expressed by many solid and liquid tumors. Methods: IGN004 was evaluated against a panel of human non-small cell lung cancer (NSCLC), melanoma, multiple myeloma (MM), and acute myeloid leukemia (AML) cell lines. Antigen expression was assessed by flow cytometry and immunohistochemistry (IHC). Anti-proliferative activity was measured by MTS assay. MHC, costimulatory, and coinhibitory molecule expression was assessed by flow cytometry. T-cell killing of tumor cells by TALL-104 effector cells was assessed by MTS assay. Human tumor cell line and patient-derived xenografts were grown in immunodeficient mice. Results: IGN004 antibody bound to the majority of tumor cell lines investigated, including melanoma, NSCLC, AML, and MM. In addition, IGN004 antibody also bound to nearly 100% of the primary solid tumor samples tested by IHC. IGN004 induced stronger growth inhibition than the unfused antibody and IFNα. Incubation of tumor cells with IGN004 caused an increase in surface expression of MHC class I, PD-L1, and OX-40L. In an in vitro T-cell killing assay using TALL-104 cells as effectors and NSCLC cells as targets, treatment of tumor cells with IGN004 led to increased effector cell killing of tumor targets (69.2% killing without IGN004 vs. 100% killing with IGN004; p = 0.001). The enhanced T-cell killing was observed even with concentrations at the sub-picomolar level (EC50 = 0.87 pM). Importantly, IGN004 demonstrated robust in vivo efficacy against MM, AML, melanoma, and NSCLC xenografts, including patient-derived tumors. Against U266 MM xenografts, IGN004 fusion protein caused complete regression of all established tumors and achieved long-term survival in 62.5% of mice. IGN004 unfused antibody caused a significant delay in tumor progression (p=0.002 vs. vehicle) but did not achieve long-term survival (p=0003 vs. IGN004 fusion protein). When efficacy was tested against a panel of patient-derived NSCLC xenografts, IGN004 fusion protein demonstrated efficacy against 7/11 tumor models, including tumor regression in 3. Against a patient-derived xenograft model of melanoma, IGN004 unfused antibody was ineffective while IGN004 fusion protein inhibited the growth of the tumors. In a patient-derived xenograft model of AML, IGN004 treatment caused a reduction in the percentage of AML cells in the blood, spleen and bone marrow, compared to PBS treated animals. Conclusions: IGN004 demonstrated robust in vitro and in vivo anti-tumor activity against both solid and liquid tumors. Treatment of tumor cells with IGN004 caused an increase in surface expression of MHC class I, PD-L1, and OX-40L. IGN004 demonstrated the ability to enhance the effector T-cell-mediated killing of NSCLC cells in an in vitro assay using the TALL-104 cell line as effectors and NSCLC cell lines as targets. Against both cell line and patient-derived human xenograft tumors IGN004 had robust in vivo anti-tumor efficacy. These results support the further development of IGN004 as a potent targeted cancer immunotherapeutic agent that also has direct anti-tumor effects. Citation Format: Kristopher Steward, Michael Gresser, Raj Sachdev, Sanjay Khare. IGN004 is an antibody-interferon-alpha fusion protein against a novel tumor-associated antigen with both direct anti-tumor and immunostimulatory effects. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B182.

Circulating tumor cells (CTCs) are associated with prognosis of patients with advanced solid tumors including lung cancer and reflect characteristics of the respective primary tumor and its metastatic deposits. Assessment of CTCs is expected to improve effectiveness of anticancer therapy and to sophisticate our knowledge to cancer metastasis. We previously reported that detection of CTCs using microcavity array (MCA) system yielded the superior sensitivity than FDA-approved CellSearch system in patients with non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Here we developed the automated CTCs detection device and evaluated its performance using preclinical spike-in model and blood samples from NSCLC and SCLC patients. The feasibility study on the assessement of PD-L1 expression level was also performed. To evaluate the recovery of CTCs preclinically, NSCLC cells, H1975, A549, H441 and PC-14 were spiked into 6 mL of peripheral whole blood obtained from healthy volunteers. Then, cells were captured and immuno-stained by using automated MCA system. CTCs were defined as those positive for DAPI and cytokeratin (CK) and negative for CD45. Additionally, normal blood cells and cancer cells were distinguished according to their size. For clinical evaluation, 6 mL of peripheral whole blood was collected from 10 healthy donors and 30 advanced lung cancer patients prior to the initiation of chemotherapy. Head-to-head comparison with CellSearch system was also conducted. PD-L1 immunostaining was established in a preclinical spike-in study using NSCLC cell lines, H820, A549, H441, and H23 with varying PD-L1 expression levels and tested as an exploratory objective in a subset of patients enrolled in a clinical study. We confirmed that up to 90% of spiked-in tumor cells were recovered by the automated MCA system, suggesting that the detection sensitivity by this automated device is on par with that by previous detection procedure. Characteristics of 30 lung cancer patients in clinical study were as follows: median age 71 (range, 48 to 85); male 70%; stage III/IV 17/83%; NSCLC/SCLC 83/17%. Cells defined as CTC were detected in 2 cases out of 10 healthy volunteers, of which CTC count was 1 and 2 in 6 mL of peripheral blood, respectively. More than 2 CTCs were detected in 77% of patients with advanced lung cancer (n = 23/30) and more than 5 CTCs were detected in 50% of patients (n = 15/30) (median CTC count 5.5). Significantly more CTCs were detected by the automated MCA system than by CellSearch system. Among stage IV NSCLC patients, patients with extrathoracic metastasis tend to have more CTCs than in those without one (median CTC count, 8 versus 4, p = 0.058). No difference in CTC counts between NSCLC and SCLC was observed in this study cohort. PD-L1 expression was assessed in a subset of patients and intra-patient heterogeneity of PD-L1 staining among CTCs was observed in patients who harbor PD-L1-positive CTCs. Our results suggest that the automated MCA device for size-based enrichment and isolation of CTCs has a clinical potential for CTCs diagnosis towards precision medicine in lung cancer. This also enables us to deal with more samples owing to its automated procedure and higher throughput. Further clinical evaluation including PD-L1 expression will be performed in an expansion cohort and the biology of CTCs will be investigated utilizing this device. Citation Format: Woong Kim, Yasuhiro Koh, Hiroaki Akamatsu, Satomi Yagi, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Ryota Shibaki, Masayuki Higuchi, Hisashige Kanbara, Takashi Kikuchi, Keiichiro Akamatsu, Masanori Nakanishi, Nobuyuki Yamamoto. Evaluation of a novel automated device for size-based enrichment and isolation of CTCs in patients with advanced lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B138.

Non-small cell lung cancer (NSCLC) has markedly increased morbidity and mortality rates worldwide. Circular RNAs were shown to regulate NSCLC progression. But the underlying pathways of the circRPPH1-mediated regulation of NSCLC still need further exploration. We evaluated circRPPH1 levels in NSCLC tissues and cell lines via qRT-PCR. Moreover, using ectopic plasmid incorporation and siRNA assays, we analysed the circRPPH1-mediated regulation of cell proliferation (CP), cell migration (CM) and cell invasion (CI) in NSCLC cell lines (H1975 and A549 cells), using CCK-8, colony forming, scratch wound and transwell assays, respectively. CircRPPH1 levels were remarkably high in the NSCLC tissues and cell lines. The transfection experiments showed that circRPPH1 overexpression was able to promote CP, CM and CI of NSCLC cells, while CP, CM and CI were significantly restrained by the knockdown of circRPPH1. We also displayed that circRPPH1 knockdown suppressed the cell progression via inactivating the PI3K/AKT and JAK2/STAT3 signalling axes. Subsequently, in vivo experiment in nude mice was demonstrated that the inhibition of circRPPH1 could reduce the tumour growth of NSCLC. circRPPH1 may accelerate the growth and metastasis of NSCLC, in culture conditions and in animal models, by stimulating the PI3K/AKT and JAK2/STAT3 signalling axes, thus promoting the development of NSCLC.