Metastatic Non-small Cell Lung Cancer Cells Research Articles

Abstract 1718: The lysine demethylase KDM2A regulates tumor cell clustering to potentiate metastasis

Abstract Approximately 70% of patients diagnosed with non-small cell lung cancer (NSCLC) are not eligible for curative resection, often due to disseminated disease at diagnosis. Thus, the advent of novel therapeutics, especially those efficacious for metastatic disease, is of primary importance. Metastasis correlates with epigenetic alterations that drive tumor plasticity and adaptive heterogeneity in response to the multiple microenvironments metastatic cells see, yet the molecular mechanisms underlying these epigenetic alterations remain poorly understood. We performed a functional genomic screen and identified the lysine 36 on histone H3 (H3K36) demethylase KDM2A as a mediator of in vivo and in vitro metastatic competence in multiple models of NSCLC. KDM2A depletion in highly metastatic NSCLC cell lines reduces their capacity to form multi-tumor cell clusters. In vivo, KDM2A depletion does not impact lung orthotopic tumor cell growth or the total number of circulating tumor cells (CTCs) but does reduce their ability to colonize distant metastatic sites. This is consistent with the fact that human CTCs found in clusters are more metastatic than CTCs which disseminate as single-cells. Because the molecular and biological consequences of H3K36 regulation are context dependent, we performed for the first time an integrated transcriptomic-epigenomic analysis of target gene regulation, KDM2A genomic binding, H3K36 methylation, and H3K27 acetylation, in metastatic cell clusters. KDM2A enhances the expression of cell adhesion and anti-oxidant genes, while suppressing inflammatory gene responses, and this regulation preferentially occurs in tumor cell clusters. Transcriptomic analysis of CTCs from patients confirms that KDM2A regulated transcriptional responses correlate with metastatic relapse across multiple cancer types. Moreover, KDM2A may function both as a transcriptional activator and repressor in metastatic clusters. As KDM2A encodes for several domains which can independently regulate E3-ubiqutin ligation, H3K36me2 demethylation, and DNA methylation, we have also performed a structure function analysis of K2MDA in the context of metastatic phenotypes. Thus, the distinct biochemical functions of KDM2A in relation to its regulation of CTC gene transcription and histone modification will also be presented. In conclusion, we have identified KDM2A as a novel regulator of NSCLC metastatic spread. The ability to inhibit KDM2A with small molecule inhibitors may be translated into effective therapeutic interventions for NSCLC patients with metastasis disease. Citation Format: Carolyn Kravitz, Kiran D. Patel, Wesley L. Cai, Anna Arnal-Estapé, Dejian Zhao, Emily Wingrove, Laura Stevens, Thomas F. Westbrook, Qin Yan, Don X. Nguyen. The lysine demethylase KDM2A regulates tumor cell clustering to potentiate metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1718.

Non–small cell lung cancer-derived exosomes promote proliferation, phagocytosis, and secretion of microglia via exosomal microRNA in the metastatic microenvironment

Non–small cell lung cancer (NSCLC) is the most common tumor that metastasizes to the brain. It is now accepted that the successful colonization and growth of tumor cells are determined by the interaction between tumor cells and the tumor microenvironment (TME). Microglia, brain innate immune cells, have been reported to play a vital role in the establishment of brain metastases. As essential mediators of intercellular communications, tumor-derived exosomes have an important role in the pathogenesis and progression of cancer by transferring their cargos to specific recipient cells. The crosstalk between microglia and tumor-derived exosomes has been extensively described. However, it is still unclear whether metastatic NSCLC cells secret exosomes to microglia and regulate the microglial functions. Here, our results showed that microglia aggregated in the brain metastatic sites. Meanwhile, microglia could take up the exosomes derived from NSCLC cells, leading to alterations of microglial morphology and increased proliferation, phagocytosis, and release of inflammatory cytokines including interleukin-6, interleukin-8, and CXCL1. Further investigation indicated that miR1246 was the most enriched microRNA in NSCLC-derived exosomes and mediated the partial effects of exosomes on microglia. Notably, miR1246 was also upregulated in the plasmatic exosomes of NSCLC patients. These results offer a new insight into the impact of NSCLC-derived exosomes on microglia and provide a new potential biomarker for diagnosing NSCLC.

Five‐to‐Seven Carbon Glycols Severely Impair Bioenergetics and Metabolism of Aggressive Lung Cancer Cells

Lung cancer is the leading cause of cancer deaths. Nonsmall cell lung cancer (NSCLC) accounts for the vast majority of the histological subtypes and at advanced stage exhibits extremely low survival rate. Certain compounds, such as glycols, inhibit migration and decreased viability of the highly metastatic NSCLC cells A549_3R has been recently shown. The glycols-induced effects are associated with a parallel reduction of ATP production, detected in A549_3R spheroids embedded in 3D desmoplastic-like extracellular matrix, following overnight treatment. Herein, selected glycols are tested for their ability to acutely compromise bioenergetics, metabolism, and viability of A549_3R cells. Therefore, 1,5-pentanediol (1,5-PD), 1,6-hexanediol (1,6-HD), and 1,7-heptanediol (1,7-Hept) turn out to be the most effective. They interfere with key enzymes in glycolysis and elicit most or all of the following responses: acute collapse of the critical mitochondrial membrane potential, substantial reduction of ATP, pyruvate and L-lactate levels, and massive increase in reactive oxygen species production. Moreover, computational docking analysis reveals that the glycols bind to the substrate-binding residues of lactate dehydrogenase A, which catalyzes the interconversion of pyruvate and lactate and is upregulated in aggressive tumors. Hence, the effective glycols may be promising anticancer compounds, or lead substances, for localized and intratumoral applications.

Exosomal lncRNA LINC01356 Derived From Brain Metastatic Nonsmall-Cell Lung Cancer Cells Remodels the Blood-Brain Barrier.

Brain metastasis is a severe complication that affects the survival of lung cancer patients. However, the mechanism of brain metastasis in lung cancer remains unclear. In this study, we constructed an in vitro BBB model and found that cells from the high-metastatic nonsmall cell lung cancer (NSCLC) cell line H1299 showed a higher capacity to pass through the blood–brain barrier (BBB), as verified by Transwell assays, than cells from the low-metastatic NSCLC cell line A549. Brain microvascular endothelial cells (BMECs) could internalize H1299-derived exosomes, which remarkably promoted A549 cells across the BBB. The BBB-associated exosomal long noncoding RNA (lncRNA) was selected from the RNA-Seq dataset (GSE126548) and verified by real-time PCR and Transwell assays. LncRNA LINC01356 was significantly upregulated in H1299 cells and exosomes derived from these cells compared to that of A549 cells. Moreover, LINC01356 was also upregulated in serum exosomes of patients with NSCLC with brain metastasis compared with those without metastasis. In addition, BMECs treated with LINC01356-deprived exosomes expressed higher junction proteins than those treated with the control exosomes, and silencing LINC01356 in exosomes derived from H1299 cells could inhibit A549 cells from crossing the BBB. These data might indicate that the exosomal lncRNA LINC01356 derived from brain metastatic NSCLC cells plays a key role in remodeling the BBB system, thereby participating in brain metastasis in lung cancer.

Soft coral-derived Aspernolide A suppressed non-small cell lung cancer induced osteolytic bone invasion via the c-Fos/NFATC1 signaling pathway

Backgroundthe incidence of distant metastases is over 30% in advanced non-small cell lung cancer (NSCLC) patients. In particular, bone is reported as the most common site of distant metastasis NSCLC. Bone metastases (BM) have a consequence of serious skeletal-related events (SREs) leading to the reduced overall survival (OS) and quality of life of NSCLC patients. Inhibition of osteolytic lesions and regulation crosstalk between metastatic NSCLC cells and bone microenvironment are the key to treating NSCLC. Due to the lack of effective treatments against NSCLC with bone metastasis, screening and identification of novel agents against both NSCLC and osteoclast effects are critically needed.MethodsWe assessed the effects of Aspernolide A (AA) on osteolysis and RANKL-induced pathways activation, bone resorption and F-actin ring formation in vitro. We evaluated AA effects on NCI-H460 and A549 cells in vitro through wound healing assay and transwell assay. Furthermore, we assessed the effects of AA in vivo using an intratibial xenograft NSCLC nude mouse model, followed by micro-computed tomography(micro-CT) and TRAcP staining.Resultsin our study, AA, a soft coral-derived agent, was shown to inhibit osteoclastogenesis via suppression of nuclear factor (NF)-κBp65, ERK, AKT and P38 phosphorylation, and then suppress the RANKL-induced c-Fos and NFATc1 activities in bone marrow macrophages (BMMs). Furthermore, AA reduced the migration and invasion of NSCLC cells through diminishing the expression of MMP9, MMP7, and N-cadherin proteins and upregulating E-cadherin expression in vitro, as well as inhibited the phosphorylation of ERK, AKT, P38, and NF-κBp65. It was also demonstrated that administration of AA could help prevent NSCLC-induced bone destruction by attenuating NSCLC development and osteoclast activity in vivo.Conclusionscollectively, these findings indicated that Aspernolide A is a potential candidate for NSCLC-induced osteolytic bone destruction.

Corrigendum] Ski prevents TGF‑β‑induced EMT and cell invasion by repressing SMAD‑dependent signaling in non‑small cell lung cancer.

Following the publication of the above paper, an interested reader drew to our attention apparent anomalies associated with Figs.2, 3 and 4; essentially, these three figures contained panels exhibiting overlapping data, such that data purportedly relating to different experiments were apparently drawn from the same original sources. [Specifically, the Ski, +TGF‑β1 data panel in Fig.2B, the Mock, +TGF‑β1 data panel in Fig.3A, and the +TGF‑β1, +SIS3 data panel in Fig.4B in the original figures were chosen incorrectly.] Upon investigating this matter with the authors, the authors have realized that they made errors in the compilation of the affected figures. The errors were made inadvertently, and the authors have been able to identify the correct data for each of the figures concerned. The corrected versions of these figures are shown opposite and on the next page. Note that these errors did not affect the overall conclusions reported in the study. The authors are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this Corrigendum; furthermore, the authors apologize for any inconvenience caused to the readership of the Journal. [the original article was published in Oncology Reports 34: 87-94, 2015; DOI: 10.3892/or.2015.3961].

MiR-182 inhibits the epithelial to mesenchymal transition and metastasis of lung cancer cells by targeting the Met gene.

The microRNA miR-182, belonging to the miR-183 family, is one of the most frequently studied cancer-related oncogenic miRNAs that is dysregulated in various cancer tissues, and it plays a crucial role in tumorigenesis and tumor progression. Studies revealed that miR-182 might function as an oncogenic or tumor suppressor miRNA in different tissues. However, the role of miR-182 in the development of lung cancer remains largely unknown. miR-182 expression in tumor samples from 58 patients, normal lung tissue samples, and lung cancer cell lines were evaluated by qRT-PCR. Survival curves were analyzed using the Kaplan-Meier method and compared with a log-rank test. Our study demonstrated that miR-182 is frequently downregulated in metastatic NSCLC cells compared with primary tumor tissues. Over-expression of miR-182 significantly inhibited the migration and invasion of lung cancer cells and promoted the expression of the epithelial marker (E-cadherin) in addition to reducing the levels of Snail in lung cancer cells. Further studies demonstrated that miR-182 negatively regulated Met via direct binding to the Met 3'-untranslated region (3'-UTR). Furthermore, we found that miR-182 suppressed the phosphorylation of AKT and the nuclear accumulation of Snail, a transcription factor that promotes the epidermal to mesenchymal transition (EMT). Moreover, miR-182 could repress cell migration, invasion, and EMT of lung cancer cells induced by hepatocyte growth factor (HGF). miR-182 might suppress the EMT and metastasis via inactivation of Met/AKT/Snail in non-small cell lung cancer (NSCLC) cells, which implicates miR-182 may be useful as a new therapeutic target in NSCLC.

CD15s/CD62E Interaction Mediates the Adhesion of Non-Small Cell Lung Cancer Cells on Brain Endothelial Cells: Implications for Cerebral Metastasis.

Expression of the cell adhesion molecule (CAM), Sialyl Lewis X (CD15s) correlates with cancer metastasis, while expression of E-selectin (CD62E) is stimulated by TNF-α. CD15s/CD62E interaction plays a key role in the homing process of circulating leukocytes. We investigated the heterophilic interaction of CD15s and CD62E in brain metastasis-related cancer cell adhesion. CD15s and CD62E were characterised in human brain endothelium (hCMEC/D3), primary non-small cell lung cancer (NSCLC) (COR-L105 and A549) and metastatic NSCLC (SEBTA-001 and NCI-H1299) using immunocytochemistry, Western blotting, flow cytometry and immunohistochemistry in human brain tissue sections. TNF-α (25 pg/mL) stimulated extracellular expression of CD62E while adhesion assays, under both static and physiological flow live-cell conditions, explored the effect of CD15s-mAb immunoblocking on adhesion of cancer cell–brain endothelium. CD15s was faintly expressed on hCMEC/D3, while high levels were observed on primary NSCLC cells with expression highest on metastatic NSCLC cells (p < 0.001). CD62E was highly expressed on hCMEC/D3 cells activated with TNF-α, with lower levels on primary and metastatic NSCLC cells. CD15s and CD62E were expressed on lung metastatic brain biopsies. CD15s/CD62E interaction was localised at adhesion sites of cancer cell–brain endothelium. CD15s immunoblocking significantly decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (p < 0.001), highlighting the role of CD15s–CD62E interaction in brain metastasis.

Amino acid transporter SLC38A3 promotes metastasis of non-small cell lung cancer cells by activating PDK1

BackgroundTumor metastasis is a finely-tuned pathological process coupled to metabolic reprogramming that includes both glutamine and glucose. The solute carrier SLC38A3, a member of amino acid/polyamine/organocation (APC) superfamily, is an l-glutamine transporter. It is not clear whether SLC38A3 involves the metastasis of NSCLC (non small cell lung cancer). MethodsThe scratch test and transwell assay were used to determine the ability of NSCLC to migrate. Cellular amino acids content was determined by mass spectrometry. The cellular response to glutamine/histidine deficiency was evaluated in A549 cells. The expression of SLC38A3 was assayed in clinical NSCLC and paratumor tissues by histoimmunochemistry staining. A nude mouse model of NSCLC metastasis was developed by tail vein injection of tumor cells. ResultsSLC38A3 was upregulated in metastatic NSCLC cells and its expression was correlated with prognosis of NSCLC patients. SLC38A3 overexpression promoted epithelial – mesenchymal transition (EMT) and migration of HCC827 and A549 human lung adenocarcinoma cells, and accelerated tumor metastasis in mice. We found that SLC38A3 decreased the cellular concentrations of glutamine and histidine, and the deficiency of glutamine or histidine activated PDK1/AKT signaling that in turn, triggered NSCLC metastasis. ConclusionsSLC38A3 activated PDK1/AKT signaling and promoted metastasis of NSCLC through regulating glutamine and histidine transport, suggesting SLC38A3 as a potential therapeutic target for treatment of NSCLC.

PP64. CD15S/CD62E HETEROPHILIC INTERACTION MEDIATES ADHESION OF NON-SMALL CELL LUNG CANCER CELLS TO BRAIN ENDOTHELIUM IN LUNG-BRAIN METASTASIS

AbstractINTRODUCTION: More than 50% of patients with lung cancer, including non-small cell lung cancer (NSCLC), will develop brain metastasis which ultimately results in a dismal prognosis. While there are several key steps in the metastastic process, we have focused on the stage where circulating cancer cells adhere to brain endothelium and extravasate through the Blood Brain Barrier (BBB). Sialyl Lewis x, also known as CD15s, is a cell adhesion molecule which is involved in lymphocyte homing and is overexpressed in non-central nervous system cancers. The purpose of our study was to investigate and characterise the interaction of CD15s and TNF-α induced CD62E (E-selectin) in brain metastatic lung cancer cells and brain derived endothelial cells. METHODS: Expression of CD62E and CD15s were evaluated in brain microvascular endothelial cells (hCMEC/D3), primary (A549 and COR-L105) and metastatic NSCLC cells (NCI-H1299, SEBTA-001 and SEBTA-005) using flow cytometry (FC), immunocytochemistry (ICC), immunohistochemistry (IHC) and Western blotting (WB). Qualitative and quantitative adhesion assays adhesion assays under static and physiological flow at shear stress 2.5 dyn/cm2 and volumetric flow rate 10mL/hr were conducted. Immunoblocking of CD15s was performed to investigate its role in adhesion on hCMEC/D3. RESULTS: While CD15s expression on brain endothelial cells was low, CD15s protein expression was detected on primary NSCLC cells, the highest CD15s expression was observed on metastatic NSCLC cells (P<0.001). CD62E expression was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC followed by primary NSCLC cells. Both CD15s and CD62E expression were detected in tissue sections of lung metastatic brain biopsies. Metastatic NSCLC adhered most strongly to hCMEC/D3 compared to primary NSCLC cells. CD15s immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (p<0.001). CONCLUSIONS: This study provides information on the role of CD15s in adhesion of NSCLC cells to brain derived endothelial cells. Our findings suggest that CD15s is involved in the increase in adhesion of circulating lung cancer cells to endothelial cells. Our findings suggests CD15s plays a crucial role in adhesion in concert with TNF-α activation and its binding partner CD62E.

MiR-150 promotes cellular metastasis in non-small cell lung cancer by targeting FOXO4.

Previous studies have shown that dysregulation of microRNA-150 (miR-150) is associated with aberrant proliferation of human non-small cell lung cancer (NSCLC) cells. However, whether miR-150 has a critical role in NSCLC cell metastasis is unknown. Here, we reveal that the critical pro-metastatic role of miR-150 in the regulation of epithelial-mesenchymal-transition (EMT) through down-regulation of FOXO4 in NSCLC. In vitro, miR-150 targets 3′UTR region of FOXO4 mRNA, thereby negatively regulating its expression. Clinically, the expression of miR-150 was frequently up-regulated in metastatic NSCLC cell lines and clinical specimens. Contrarily, FOXO4 was frequently down-regulated in NSCLC cell lines and clinical specimens. Functional studies show that ectopic expression of miR-150 enhanced tumor cell metastasis in vitro and in a mouse xenograft model, and triggered EMT-like changes in NSCLC cells (including E-cadherin repression, N-cadherin and Vimentin induction, and mesenchymal morphology). Correspondingly, FOXO4 knockdown exhibited pro-metastatic and molecular effects resembling the effect of miR-150 over-expression. Moreover, NF-κB/snail/YY1/RKIP circuitry regulated by FOXO4 were likely involved in miR-150-induced EMT event. Simultaneous knockdown of miR-150 and FOXO4 abolished the phenotypic and molecular effects caused by individual knockdown of miR-150. Therefore, our study provides previously unidentified pro-metastatic roles and mechanisms of miR-150 in NSCLC.

Abstract 1584: Musashi-2 (MSI2) drives TGFBR1/SMAD3 dependent partial EMT and supports VEGFR2 expression and metastasis of human and mouse NSCLC cells

Abstract About 221,200 new patients will be diagnosed with lung cancer and ∼158,040 will succumb to this disease in the United States in 2015. Non-small cell lung cancer (NSCLC) has a 17.4% overall 5-year survival, with metastasis contributing to the vast majority of deaths. Analyzing NSCLC tumors spontaneously arising in KrasLA1/+; P53R172HG/+ (KP) mice, we identified Musashi-2 (MSI2) protein, a stem cell-associated factor that is regulates mRNA translation, as upregulated in the metastasis-competent mouse cell lines. Importantly, MSI2 shRNA depletion in either mouse or human NSCLC cells decreased invasion in Matrigel in vitro and decreased metastasis upon orthotopic injection 129Sv immunocompetent mice in vivo. Mechanistically, by both overexpressing Msi2 cDNA in 393p murine NSCLC and shRNA depleting MSI2 in four independent mouse and human NSCLC cell lines, we defined MSI2 as a driver of a partial epithelial-mesenchymal transition (EMT) program in NSCLC cells. In support of EMT, MSI2 increases the expression of the Snail and Slug pro-EMT transcription factors, as well as the mesenchymal protein vimentin (VMN). MSI2 also downregulates expression of the extracellular matrix component fibronectin (FN1) and tight junction proteins Claudin-3, 5 and 7. However, MSI2 also inhibits protein expression of Zeb-1 and Zeb-2, while sustaining expression of E-cadherin (E-Cad), associated with epithelial identity. Moreover, MSI2 represses NOTCH-1 and upregulates VEGFR2 at the mRNA and protein levels. This is of interest, as NOTCH-1 has been shown to regulate VEGFR2 in angiogenic signaling. We found that knockdown of NOTCH-1 in MSI2-depleted mouse and human NSCLC cells rescued the loss of VEGFR2 expression, suggesting MSI2 increases VEGFR2 expression in a NOTCH-1 dependent manner. Additionally, siRNA of VEGFR2 in the highly metastatic NSCLC cell line 344sq significantly decreased Matrigel invasion, but had only a limited effect in 344sq derivative lines with stable depletion of MSI2, and human NSCLC experiments are ongoing. Together, these results indicate a possible role of MSI2/NOTCH-1/VEGFR2 axis in NSCLC, and suggest that MSI2 connects cancer cell stemness, EMT, and angiogenesis in lung cancer metastasis. Citation Format: Alexander Kudinov, Alexander Deneka, Anna Nikonova, Ilya Serebriiskii, Tim N. Beck, Qi Cai, Brian L. Egleston, Emmanuelle Nicolas, Hossein Borghaei, Don Gibbons, Jonathan Kurie, Erica A. Golemis, Yanis Boumber. Musashi-2 (MSI2) drives TGFBR1/SMAD3 dependent partial EMT and supports VEGFR2 expression and metastasis of human and mouse NSCLC cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1584.

Musashi-2 (MSI2) supports TGF-β signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis

Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of Kras(LA1/+);P53(R172HΔG/+) (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial-mesenchymal transition, including the TGF-β receptor 1 (TGFβR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFβRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial-mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFβR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness.

TNF-α enhancement of CD62E mediates adhesion of non-small cell lung cancer cells to brain endothelium via CD15 in lung-brain metastasis.

BackgroundCD15, which is overexpressed on various cancers, has been reported as a cell adhesion molecule that plays a key role in non-CNS metastasis. However, the role of CD15 in brain metastasis is largely unexplored. This study provides a better understanding of CD15/CD62E interaction, enhanced by tumor necrosis factor-α (TNF-α), and its correlation with brain metastasis in non–small cell lung cancer (NSCLC).MethodsCD15 and E-selectin (CD62E) expression was demonstrated in both human primary and metastatic NSCLC cells using flow cytometry, immunofluorescence, and Western blotting. The role of CD15 was investigated using an adhesion assay under static and physiological flow live-cell conditions. Human tissue sections were examined using immunohistochemistry.ResultsCD15, which was weakly expressed on hCMEC/D3 human brain endothelial cells, was expressed at high levels on metastatic NSCLC cells (NCI-H1299, SEBTA-001, and SEBTA-005) and at lower levels on primary NSCLC (COR-L105 and A549) cells (P < .001). The highest expression of CD62E was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC cells followed by primary NSCLC cells. Metastatic NSCLC cells adhered most strongly to hCMEC/D3 compared with primary NSCLC cells. CD15 immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (P < .0001), confirming a correlation between CD15 and cerebral metastasis. Both CD15 and CD62E expression were detected in lung metastatic brain biopsies.ConclusionThis study enhances the understanding of cancer cell-brain endothelial adhesion and confirms that CD15 plays a crucial role in adhesion in concert with TNF-α activation of its binding partner, CD62E.

Abstract 4098: Musashi-2 (MSI2) activates TGF-β signaling and inhibits CLDN7 to promote non-small cell lung cancer (NSCLC) metastasis

Abstract Approximately 220,000 new patients were diagnosed with lung cancer and ∼160,000 died from this disease in the United States in 2014. Non-small cell lung cancer (NSCLC) has a 5 year survival rate of approximately 16%, with most deaths associated with distant metastasis. To gain a better insight into the regulation of metastasis, we studied metastasis-competent versus low metastasis-potential cell lines derived from NSCLC tumors of KrasLA1/+; P53R172HG/+ (KP) mice. Initial screening showed consistent and statistically significant upregulation of Musashi-2 (MSI2) in highly metastatic cells on both mRNA and protein level. Furthermore, we established statistically significant elevation of MSI2 protein expression in 123 human NSCLC tumor specimens versus normal lung tissue (p&amp;lt;0.0001). MSI2 is an RNA binding protein that regulates mRNA translation and is upregulated and functionally important in hematologic malignancies including CML and AML. MSI2 knockdown in four independent metastatic murine and human NSCLC cell lines very significantly decreased invasion in vitro but did not significantly change cell proliferation or survival. In orthotropic lung injection of mouse NSCLC cells into the lungs of immunocompetent 129Sv mice, MSI2 depletion dramatically decreased invasion of mediastinal lymph nodes and abrogated metastasis. Reverse-phase protein array (RPPA) screening indicated that Msi2 depletion significantly affected the expression of multiple proteins associated with epithelial-mesenchymal transition (EMT) and control of cell-cell attachments, including the extracellular matrix component fibronectin (FN1) and the tight junction protein claudin-7 (CLDN7). These and additional targets were validated in all four NSCLC model systems. MSI2 dependent control of FN1 expression was mediated at the level of mRNA induction, while CLDN7 expression was strikingly upregulated (10-fold) at the protein but not mRNA level, nominating CLDN7 as a putative direct MSI2 target. Parallel evaluation of a series of candidate direct MSI2 translational targets indicated MSI2 depletion downregulated translation of TGF-β receptor (TGF-βRI) and SMAD3. Morphologically, MSI2 depleted cells were marked by a greater degree of cell-cell attachment, potentially explaining their decreased invasive capacity. Based on this work, we propose that MSI2 supports TGF-βRI dependent EMT signaling and downregulates tight junction controls in a subset of metastatic NSCLC, and may influence tumor response to inhibitors targeting the TGF-β pathway. Citation Format: Alexander Kudinov, Alexander Deneka Deneka, Anna Nikonova, Young-Ho Ahn, Xin Liu Liu, Ilya Serebriiskii, Andrey Efimov, Dong-Hua Yang, Mark Andrake Andrake, Emmanuelle Nicolas, Brian Egleston, Hossein Borghaei, Don Gibbons, Jonathan Kurie, Erica Golemis, Yanis Boumber. Musashi-2 (MSI2) activates TGF-β signaling and inhibits CLDN7 to promote non-small cell lung cancer (NSCLC) metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4098. doi:10.1158/1538-7445.AM2015-4098

Ski prevents TGF-β-induced EMT and cell invasion by repressing SMAD-dependent signaling in non-small cell lung cancer.

Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis, which confers cancer cells with increased motility and invasiveness, and EMT is characterized by loss of epithelial marker E-cadherin and gain of mesenchymal marker N-cadherin. Transforming growth factor-β (TGF-β) signaling is a crucial inducer of EMT in various types of cancer. Ski is an important negative regulator of TGF-β signaling, which interacts with SMADs to repress TGF-β signaling activity. Although there is accumulating evidence that Ski functions as a promoter or suppressor in human types of cancer, the molecular mechanisms by which Ski affects TGF-β-induced EMT and invasion in non-small cell lung cancer (NSCLC) are not largely elucidated. In the present study, we investigated the mechanistic role of Ski in NSCLC metastasis. Ski was significantly reduced in metastatic NSCLC cells or tissues when compared with non-metastatic NSCLC cells or tissues. Moreover, following TGF-β stimulation Ski-silenced A549 cells had more significant features of EMT and a higher invasive activity when compared with A549 cells overexpressing Ski. Mechanistically, Ski-silenced and overexpressed A549 cells showed an increase and a reduction in the SMAD3 phosphorylation level, respectively. This was supported by plasminogen activator inhibitor-1 (PAI-1) promoter activity obtained in Ski-silenced and overexpressed A549 cells. However, after treatment of SIS3 (inhibitor of SMAD3 phosphorylation) followed by TGF-β1 stimulation, we did not observe any effect of Ski on TGF-β-induced EMT, and invasion in Ski-silenced and overexpressed A549 cells. In conclusion, our findings suggest that Ski represses TGF-β-induced EMT and invasion by inhibiting SMAD-dependent signaling in NSCLC.

Photodynamic Therapy for Lung Cancer and Malignant Pleural Mesothelioma

Photodynamic therapy (PDT) is a form of non-ionizing radiation therapy that uses a drug, called a photosensitizer, combined with light to produce singlet oxygen ((1)O2) that can exert anti-cancer activity through apoptotic, necrotic, or autophagic tumor cell death. PDT is increasingly being used to treat thoracic malignancies. For early-stage non-small cell lung cancer (NSCLC), PDT is primarily employed as an endobronchial therapy to definitively treat endobronchial or roentgenographically occult tumors. Similarly, patients with multiple primary lung cancers may be definitively treated with PDT. For advanced or metastatic NSCLC and small cell lung cancer (SCLC), PDT is primarily employed to palliate symptoms from obstructing endobronchial lesions causing airway compromise or hemoptysis. PDT can be used in advanced NSCLC to attempt to increase operability or to reduce the extent of operation intervention required, and selectively to treat pleural dissemination intraoperatively following macroscopically complete surgical resection. Intraoperative PDT can be safely combined with macroscopically complete surgical resection and other treatment modalities for malignant pleural mesothelioma (MPM) to improve local control and prolong survival. This report reviews the mechanism of and rationale for using PDT to treat thoracic malignancies, details prospective and major retrospectives studies of PDT to treat NSCLC, SCLC, and MPM, and describes improvements in and future roles and directions of PDT.

MiR-339-5p inhibits cell migration and invasion in vitro and may be associated with the tumor-node-metastasis staging and lymph node metastasis of non-small cell lung cancer.

The aim of the present study was to investigate the potential role of microRNA (miRNA or miR) in invasion and metastasis of non-small cell lung cancer (NSCLC). miRNA-microarray analysis was used to detect the differentially expressed miRNAs between various metastatic levels of NSCLC cells. The microarray results were verified by quantitative polymerase chain reaction. The most clearly altered miRNA, miR-339-5p, was transfected into NSCLC cells and cell migration and invasion were investigated. The expression of miR-339-5p was 3.4662-fold higher in the lower metastatic NSCLC cells. miR-339-5p significantly decreased tumor-cell migration and the invasion capacity in vitro. In conclusion, miR-339-5p is important in NSCLC invasion and metastasis, indicating that miR-339-5p could be further evaluated as a biomarker for predicting the survival time of patients with NSCLC.

The microRNA-200 family targets multiple non-small cell lung cancer prognostic markers in H1299 cells and BEAS-2B cells

Lung cancer remains the leading cause of cancer-related mortality for both men and women. Tumor recurrence and metastasis is the major cause of lung cancer treatment failure and death. The microRNA-200 (miR-200) family is a powerful regulator of the epithelial-mesenchymal transition (EMT) process, which is essential in tumor metastasis. Nevertheless, miR-200 family target genes that promote metastasis in non-small cell lung cancer (NSCLC) remain largely unknown. Here, we sought to investigate whether the microRNA-200 family regulates our previously identified NSCLC prognostic marker genes associated with metastasis, as potential molecular targets. Novel miRNA targets were predicted using bioinformatics tools based on correlation analyses of miRNA and mRNA expression in 57 squamous cell lung cancer tumor samples. The predicted target genes were validated with quantitative RT-PCR assays and western blot analysis following re-expression of miR-200a, -200b and -200c in the metastatic NSCLC H1299 cell line. The results show that restoring miR-200a or miR-200c in H1299 cells induces downregulation of DLC1, ATRX and HFE. Reinforced miR-200b expression results in downregulation of DLC1, HNRNPA3 and HFE. Additionally, miR-200 family downregulates HNRNPR3, HFE and ATRX in BEAS-2B immortalized lung epithelial cells in quantitative RT-PCR and western blot assays. The miR-200 family and these potential targets are functionally involved in canonical pathways of immune response, molecular mechanisms of cancer, metastasis signaling, cell-cell communication, proliferation and DNA repair in Ingenuity pathway analysis (IPA). These results indicate that re-expression of miR-200 downregulates our previously identified NSCLC prognostic biomarkers in metastatic NSCLC cells. These results provide new insights into miR-200 regulation in lung cancer metastasis and consequent clinical outcome, and may provide a potential basis for innovative therapeutic approaches for the treatment of this deadly disease.

Osteopontin knockdown suppresses non-small cell lung cancer cell invasion and metastasis.

Osteopontin (OPN) was identified as one of the leading genes that promote the metastasis of malignant tumor. However, the mechanism by which OPN mediates metastasis in non-small cell lung cancer (NSCLC) remains unknown. The aim of the study is to investigate the biological significance and the related molecular mechanism of OPN expression in lung cancer cell line. Lentiviral-mediated RNA interference was applied to inhibit OPN expression in metastatic human NSCLC cell line (A549). The invasion, proliferation, and metastasis were evaluated OPN-silenced in A549 cells in vitro and in vivo. The related mechanism was further investigated. Interestingly, OPN knockdown significantly suppressed the invasiveness of A549 cells, but had only a minor effect on the cellular migration and proliferation. Moreover, we demonstrated that OPN knockdown significantly reduced the levels of matrix metalloproteinase (MMP)-2 and urokinase plasminogen activator (uPA), and led to an obvious inhibition of both in vitro invasion and in vivo lung metastasis of A549 cells (P < 0.001). Our data demonstrate that OPN contributes to A549 cell metastasis by stimulating cell invasion, independent of cellular migration and proliferation. OPN could be a new treatment target of NSCLC.