Non-small Cell Lung Carcinoma Cells Research Articles

Demethoxycurcumin-Loaded Chitosan Nanoparticle Downregulates DNA Repair Pathway to Improve Cisplatin-Induced Apoptosis in Non-Small Cell Lung Cancer.

Demethoxycurcumin (DMC), through a self-assembled amphiphilic carbomethyl-hexanoyl chitosan (CHC) nanomatrix has been successfully developed and used as a therapeutic approach to inhibit cisplatin-induced drug resistance by suppressing excision repair cross-complementary 1 (ERCC1) in non-small cell lung carcinoma cells (NSCLC). Previously, DMC significantly inhibited on-target cisplatin resistance protein, ERCC1, via PI3K-Akt-snail pathways in NSCLC. However, low water solubility and bioavailability of DMC causes systemic elimination and prevents its clinical application. To increase its bioavailability and targeting capacity toward cancer cells, a DMC-polyvinylpyrrolidone core phase was prepared, followed by encapsulating in a CHC shell to form a DMC-loaded core-shell hydrogel nanoparticles (DMC-CHC NPs). We aimed to understand whether DMC-CHC NPs efficiently potentiate cisplatin-induced apoptosis through downregulation of ERCC1 in NSCLC. DMC-CHC NPs displayed good cellular uptake efficiency. Dissolved in water, DMC-CHC NPs showed comparable cytotoxic potency with free DMC (dissolved in DMSO). A sulforhodamine B (SRB) assay indicated that DMC-CHC NPs significantly increased cisplatin-induced cytotoxicity by highly efficient intracellular delivery of the encapsulated DMC. A combination of DMC-CHC NPs and cisplatin significantly inhibited on-target cisplatin resistance protein, ERCC1, via the PI3K-Akt pathway. Also, this combination treatment markedly increased the post-target cisplatin resistance pathway including bax, and cytochrome c expressions. Thymidine phosphorylase (TP), a main role of the pyrimidine salvage pathway, was also highly inhibited by the combination treatment. The results suggested that enhancement of the cytotoxicity to cisplatin via administration of DMC-CHC NPs was mediated by down-regulation of the expression of TP, and ERCC1, regulated via the PI3K-Akt pathway.

Self-targeted knockdown of CD44 improves cisplatin sensitivity of chemoresistant non-small cell lung cancer cells.

Chemoresistance remains a major challenge for effective chemotherapy of non-small-cell lung carcinoma (NSCLC). CD44 expression is related to the susceptibility of various cancer cell types to anticancer drugs. Here, we systematically investigated the CD44-dependent chemoresistance of NSCLC cells and developed a liposomal siRNA delivery system to overcome this chemoresistance by the self-targeted downregulation of CD44. We confirmed the relationship between the expression of CD44 and the chemosensitivity of NSCLC cells using flow cytometry and MTT assay. We then generated and characterized cisplatin-resistant cell lines and compared the expression of CD44 in resistant cells to that in parental cells using western blotting. To evaluate whether the chemosensitivity of resistant cells depends on CD44 expression, we performed CD44 knockdown using CD44 siRNA and detected the chemosensitivity of these cells. Additionally, we prepared hyaluronic acid (HA)-coated liposomes as a targeted delivery system to selectively deliver CD44-specific siRNA to chemoresistant NSCLC cells and observed whether the chemosensitivity of these cells was improved. We found that CD44 expression is inversely proportional to the degree of cellular response to cisplatin chemotherapy and that CD44 is overexpressed in chemoresistant NSCLC cells. By performing CD44 knockdown using siRNA, we reconfirmed that the chemosensitivity of resistant cells depends on CD44 expression. We also observed that HA-liposome-mediated siRNA delivery prior to cisplatin chemotherapy significantly reduced CD44 expression and enhanced cisplatin sensitivity in chemoresistant NSCLC cells. These results suggest that self-targeted downregulation of chemoresistance-associated cell surface proteins during chemotherapy is an effective therapeutic strategy for overcoming the chemoresistance of NSCLC cells.

TMEM106A inhibits cell proliferation, migration, and induces apoptosis of lung cancer cells.

Transmembrane protein 106A (TMEM106A) has been found to function as tumor suppressor in gastric and renal cancer. However, the role of TMEM106A in nonsmall-cell lung carcinoma (NSCLC) has not been investigated. In this study, we evaluated the expression profile of TMEM106A in NSCLC tissues and cell line, and explored the roles of TMEM106A in NSCLC cell lines. Our results showed that TMEM106A expression was significantly decreased in human NSCLC tissues. In vitro assays showed that TMEM106A expression in NSCLC cell lines was much lower than that in the bronchial epithelial cell line. Besides, overexpression of TMEM106A reduced cell proliferation, migration, and invasion, while induced cell apoptosis in NSCLC cells. TMEM106A overexpression repressed epithelial-mesenchymal transition (EMT), which was illustrated by increased E-cadherin expression and decreased the expressions of N-cadherin, and vimentin. In addition, TMEM106A overexpression suppressed the activation of phosphoinositide 3-kinase/protein kinase B/nuclear factor-κB (PI3K/Akt/NF-κB) signaling pathway in NSCLC cells. Our results indicated that TMEM106A acted as a tumor suppressor in NSCLC, and could be a therapeutic target for the management of NSCLC.

Role of INSL4 Signaling in Sustaining the Growth and Viability of LKB1-Inactivated Lung Cancer.

The LKB1 tumor suppressor gene is commonly inactivated in non-small cell lung carcinomas (NSCLC), a major form of lung cancer. Targeted therapies for LKB1-inactivated lung cancer are currently unavailable. Identification of critical signaling components downstream of LKB1 inactivation has the potential to uncover rational therapeutic targets. Here we investigated the role of INSL4, a member of the insulin/IGF/relaxin superfamily, in LKB1-inactivated NSCLCs. INSL4 expression was analyzed using global transcriptome profiling, quantitative reverse transcription PCR, western blotting, enzyme-linked immunosorbent assay, and RNA in situ hybridization in human NSCLC cell lines and tumor specimens. INSL4 gene expression and clinical data from The Cancer Genome Atlas lung adenocarcinomas (n = 515) were analyzed using log-rank and Fisher exact tests. INSL4 functions were studied using short hairpin RNA (shRNA) knockdown, overexpression, transcriptome profiling, cell growth, and survival assays in vitro and in vivo. All statistical tests were two-sided. INSL4 was identified as a novel downstream target of LKB1 deficiency and its expression was induced through aberrant CRTC-CREB activation. INSL4 was highly induced in LKB1-deficient NSCLC cells (up to 543-fold) and 9 of 41 primary tumors, although undetectable in all normal tissues except the placenta. Lung adenocarcinomas from The Cancer Genome Atlas with high and low INSL4 expression (with the top 10th percentile as cutoff) showed statistically significant differences for advanced tumor stage (P < .001), lymph node metastasis (P = .001), and tumor size (P = .01). The INSL4-high group showed worse survival than the INSL4-low group (P < .001). Sustained INSL4 expression was required for the growth and viability of LKB1-inactivated NSCLC cells in vitro and in a mouse xenograft model (n = 5 mice per group). Expression profiling revealed INSL4 as a critical regulator of cell cycle, growth, and survival. LKB1 deficiency induces an autocrine INSL4 signaling that critically supports the growth and survival of lung cancer cells. Therefore, aberrant INSL4 signaling is a promising therapeutic target for LKB1-deficient lung cancers.

LncRNA PCAT6 promotes non-small cell lung cancer cell proliferation, migration and invasion through regulating miR-330-5p.

BackgroundInvestigating the roles of lncRNA prostate cancer-associated transcript 6 (PCAT6) in modulating the growth and aggressiveness of non-small-cell lung carcinoma (NSCLC) cell.MethodThe levels of PCAT6 in NSCLC tissues and cell lines were determined by quantitative real-time PCR assay. MTT as well as colony formation assays were applied to explore the effect of PCAT6 on the growth of NSCLC cell in vitro. Wound healing and Transwell assays were utilized to analyze the impact of PCAT6 on the migration and invasion of NSCLC cell. Bioinformatics analysis and luciferase reporter assay were used to prove that miR-330-5p was the target of PCAT6. Colony formation, wound healing, and Transwell invasion assays were applied to demonstrate that PCAT6 promoted NSCLC cell growth, migration, and invasion through binding miR-330-5p. Finally, xenograft model was used to explore the role of PCAT6 in the tumor growth of NSCLC cell in vivo.ResultsPCAT6 was highly overexpressed in NSCLC tissues and cells compared with normal tissues and non-tumorigenic bronchial epithelial cell line, BEAS-2B. Downregulation of PCAT6 markedly reduced the proliferation, migration, and invasion of NSCLC cell. Moreover, down-expression of PCAT6 significantly increased the level of miR-330-5p in NSCLC cell. Further functional experiments indicated that down-expression of miR-330-5p reversed the inhibitory effect of PCAT6 on NSCLC cell growth, migration, and invasion.ConclusionOur results reveal that lncRNA PCAT6 facilitates the proliferation, migration, and invasion of NSCLC cell via competitively binding to miR-330-5p.

Development of a Fluorinated Analogue of Erlotinib for PET Imaging of EGFR Mutation\u2013Positive NSCLC

PurposePositron emission tomography (PET) using [11C]erlotinib identifies non-small cell lung carcinoma (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFRm). The short half-life of C-11, however, limits its clinical utility to centers with a nearby cyclotron. We therefore developed a F-18–labeled analogue of erlotinib for imaging EGFRm NSCLC.Procedures6-O-Fluoroethylerlotinib (6-O-FEE) was synthesized and its anti-proliferative activity was tested using human NSCLC cell lines. The F-18–labeled compound, 6-O-[18F]FEE, was obtained in a two-step synthesis, and PET acquisitions were carried out following its injection to NSCLC tumor–bearing mice.ResultsIn vitro, 6-O-FEE had maintained the selectivity and potency of erlotinib to EGFRm NSCLC. In vivo, 6-O-[18F]FEE accumulation in EGFRm tumors at 60 min after injection was 2- and 3.3-fold higher than in erlotinib-resistant or erlotinib-insensitive tumors, respectively.Conclusions6-O-[18F]FEE holds promise for imaging EGFRm NSCLC, warranting further investigation to fully explore its potential for stratifying NSCLC patients.

MicroRNA-212 suppresses nonsmall lung cancer invasion and migration by regulating ubiquitin-specific protease-9.

MicroRNAs (miRNAs) play crucial roles in various biological processes, including migration, proliferation, differentiation, cell cycling, and apoptosis. Epithelial-mesenchymal transition (EMT) has been shown to be related to the capability of migration and invasion in many tumor cells. In this study, we used wound-healing assay and transwell invasion to analysis the capability of migration and invasion in non-small-cell lung carcinoma (NSCLC), respectively. The expression of ubiquitin-specific protease-9-X-linked (USP9X) and miR-212 messenger RNA (mRNA) was determined by quantitative real-time polymerase chain reaction and Western blot analysis was used to determine the E-cadherin and vimentin expression. Our results showed that miR-212 mimic inhibited cell migration and invasion, while miR-212 inhibitor increased cell migration and invasion. There was no significant difference between WP1130 and miR-212 mimic combined with WP1130 groups. Moreover, WP1130 inhibited the capability of the migration and invasion of NSCLC cells. Western blot analysis displayed that miR-212 mimic upregulated E-cadherin expression and downregulated vimentin expression, while miR-212 inhibitor downregulated E-cadherin and upregulated vimentin expression. These data showed that miR-212 regulated NSCLC cell invasion and migration by regulating USP9X expression. Taken together, these findings indicated that miR-212 regulated NSCLC cells migration and invasion through targeting USP9X involved in EMT.

Aptamer-miR-34c Conjugate Affects Cell Proliferation of Non-Small-Cell Lung Cancer Cells

MicroRNAs (miRNAs) are key regulators of different human processes that represent a new promising class of cancer therapeutics or therapeutic targets. Indeed, in several tumor types, including non-small-cell lung carcinoma (NSCLC), the deregulated expression of specific miRNAs has been implicated in cell malignancy. As expression levels of the oncosuppressor miR-34c-3p are decreased in NSCLC compared to normal lung, we show that reintroduction of miR-34c-3p reduces NSCLC cell survival in vitro. Further, in order to deliver the miR-34c-based therapeutic selectively to tumor cells, we took advantage of a reported nucleic acid aptamer (GL21.T) that binds and inhibits the AXL transmembrane receptor and is rapidly internalized in the target cells. By applying methods successfully used in our laboratory, we conjugated miR-34c to the GL21.T aptamer as targeting moiety for the selective delivery to AXL-expressing NSCLC cells. We demonstrate that miR-34c-3p and the GL21.T/miR-34c chimera affect NSCLC cell proliferation and are able to overcome acquired RTK-inhibitor resistance by targeting AXL receptor. Thus, the GL21.T/miR-34c chimera exerts dual inhibition of AXL at functional and transcriptional levels and represents a novel therapeutic tool for the treatment of NSCLC.

RIF1 promotes tumor growth and cancer stem cell-like traits in NSCLC by protein phosphatase 1-mediated activation of Wnt/\u03b2-catenin signaling

Wnt/β-catenin signaling is essential for proliferation and maintenance of cancer stem cell-like traits of various cancer cells. In non-small-cell lung carcinoma (NSCLC), the mechanisms underlying the hyperactivation of Wnt signaling remain unclear, as mutations in APC and β-catenin genes are rare in NSCLC. RIF1 has been shown upregulated in breast and cervical cancer, this study intends to find out the potential effects of the expression and biological functions of RIF1 in NSCLC. Here we revealed that RIF1 was highly expressed in NCSLC at both mRNA and protein levels. RIF1 expression was significantly associated with clinical stage (P < 0.05) and prognosis (P < 0.001) of NSCLC patients. RIF1 knockdown inhibited NSCLC cell growth in vitro and in vivo, whereas overexpression of RIF1 in NSCLC cell lines promoted cell growth, cell cycle progression and cancer stem cell (CSC)-like properties via promoting PP1–AXIN interaction and thereby activating Wnt/β-catenin signaling. Inhibition of PP1 in RIF1-overexpressed cells counteracted the effects of RIF1 on cell growth and CSC-like phenotype, as well as the Wnt/β-catenin signaling. RIF1 expression was positively correlated with β-catenin at the protein level in 32 NSCLC tissues. RIF1 expression closely related to MYC (r = 0.28, P < 0.001) and CCND1 (r = 0.14, P < 0.01) expression at the mRNA level in cohorts of The Cancer Genome Atlas (TCGA). These results indicated that RIF1 had an oncogenic role as a novel positive regulator of Wnt/β-catenin signaling by directing PP1 to dephosphorylate AXIN; this novel mechanism may present a new therapeutic target for NSCLC.

Astragalus polysaccharide combined with 10-hydroxycamptothecin inhibits metastasis in non-small cell lung carcinoma cell lines via the MAP4K3/mTOR signaling pathway.

Non-small cell lung carcinoma (NSCLC) is a life-threatening malignancy. The level of the cell growth regulator mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) has been shown to be correlated with a high risk of NSCLC recurrence and poor recurrence-free survival rate. The present study examined the effects of Astragalus polysaccharide (APS) and 10-hydroxycamptothecin (HCPT), which are associated with marked suppression and dephosphorylation of the MAP4K3/mammalian target of rapamycin (mTOR) signaling pathway, in the H1299 NSCLC cell line. APS and HCPT decreased H1299 cell viability, induced apoptosis and altered the cell cycle stages, as evaluated using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometric analysis. Furthermore, APS increased the expression of apoptosis-associated genes B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX), of proteases cysteine-aspartic acid protease (caspase)-3 and -9, and of cytochrome c. HCPT promoted autophagy in H1299 cells, with concomitant suppression of the expression of MAP4K3 and downregulation of mTOR signaling. Notably, combination treatment with the two agents reduced the migration and invasion of H1299 cells compared with the single treatments. It was also demonstrated that the overexpression of MAP4K3 promoted the migration and invasion of H1299 cells, and that the kinase activity was essential to this. These findings suggested that MAP4K3 may be an attractive target for the treatment of NSCLC

EGF-induced nuclear localization of SHCBP1 activates \u03b2-catenin signaling and promotes cancer progression

Aberrant activation of EGFR represents a common event in non-small cell lung carcinoma (NSCLC) and activates various downstream signaling pathways. While EGFR activation of β-catenin signaling was previously reported, the mediating mechanism remains unclear. Our current study found that EGFR activation in NSCLC cells releases SHC-binging protein 1 (SHCBP1) from SHC adaptor protein 1 (SHC1), which subsequently translocates into the nucleus and directly promotes the transactivating activity of β-catenin, consequently resulting in development of NSCLC cell stemness and malignant progression. Furthermore, SHCBP1 promotes β-catenin activity through enhancing the CBP/β-catenin interaction, and most interestingly, a candidate drug that blocks the CBP/β-catenin binding effectively abrogates the aforementioned biological effects of SHCBP1. Clinically, SHCBP1 level in NSCLC tumors was found to inversely correlate with patient survival. Together, our study establishes a novel convergence between EGFR and β-catenin pathways and highlights a potential significance of SHCBP1 as a prognostic biomarker and a therapeutic target.

STAT3 Cyclic Decoy Demonstrates Robust Antitumor Effects in Non-Small Cell Lung Cancer.

Constitutively activated STAT3 plays a critical role in non-small cell lung carcinoma (NSCLC) progression by mediating proliferation and survival. STAT3 activation in normal cells is transient, making it an attractive target for NSCLC therapy. The therapeutic potential of blocking STAT3 in NSCLC was assessed utilizing a decoy approach by ligating a double-stranded 15-mer oligonucleotide that corresponds to the STAT3 response element of STAT3-target genes, to produce a cyclic STAT3 decoy (CS3D). The decoy was evaluated using NSCLC cells containing either wild-type EGFR (201T) or mutant EGFR with an additional EGFRi resistance mutation (H1975). These cells are resistant to EGFR inhibitors and require an alternate therapeutic approach. CS3D activity was compared with an inactive cyclic control oligonucleotide (CS3M) that differs by a single base pair, rendering it unable to bind to STAT3 protein. Transfection of 0.3 μmol/L of CS3D caused a 50% inhibition in proliferation in 201T and H1975 cells, relative to CS3M, and a 2-fold increase in apoptotic cells. Toxicity was minimal in normal cells. CS3D treatment caused a significant reduction of mRNA and protein expression of the STAT3 target gene c-Myc and inhibited colony formation by 70%. The active decoy decreased the nuclear pool of STAT3 compared with the mutant. In a xenograft model, treatments with CS3D (5 mg/kg) caused a potent 96.5% and 81.7% reduction in tumor growth in 201T (P < 0.007) and H1975 models (P < 0.0001), respectively, and reduced c-Myc and p-STAT3 proteins. Targeting STAT3 with the cyclic decoy could be an effective therapeutic strategy for NSCLC. Mol Cancer Ther; 17(9); 1917-26. ©2018 AACR.

Demethoxycurcumin sensitizes the response of non-small cell lung cancer to cisplatin through downregulation of TP and ERCC1-related pathways

Excision repair cross-complementary 1 (ERCC1) overexpression in lung cancer cells is strongly correlated with its resistance to platinum-based chemotherapy. Overexpression of thymidine phosphorylase (TP) reverts platinum-induced cancer cell death. Curcumin has been reported to enhance antitumor properties through the suppression of TP and ERCC1 in non-small cell lung carcinoma cells (NSCLC). Nevertheless, whether two other curcuminoids, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) from Curcuma longa demonstrate antitumor activity like that of curcumin remain unknown. MTT assay was conducted to determine the cell cytotoxicity. Western blotting was used to determine the protein expressions. Docking is the virtual screening of a database of compounds and predicting the strongest binders based on various scoring functions. BIOVIA Discovery Studio 4.5 (D.S. 4.5) were used for docking. Firstly, when compared with curcumin and BDMC, DMC exhibited the most potent cytotoxic effect on NSCLC, most importantly, MRC-5, a lung fetal fibroblast, was insensitive to DMC (under 30µM). Secondly, DMC alone significantly inhibited on-target cisplatin (CDDP) resistance protein, ERCC1, via PI3K-Akt-snail pathways, and TP protein expression in A549 cells. Thirdly, DMC treatment markedly increased post-target CDDP resistance pathway including Bax and cytochrome c. DMC significantly decreased Bcl-2 protein expressions. Finally, MTT assay indicated that DMC significantly increased CDDP-induced cytotoxicity and was confirmed with an increased Bax/Bcl-2 ratio, indicating upregulation of caspase-3. We concluded that enhancement of the cytotoxicity to CDDP by coadminstration with DMC was mediated by down-regulation of the expression of TP and ERCC1, regulated by PI3K-Akt-Snail pathway inactivation.

Inhibitory Effect of MiR-449b on Cancer Cell Growth and Invasion through LGR4 in Non-Small-Cell Lung Carcinoma.

Non-small-cell lung carcinoma (NSCLC) is one of the most frequently diagnosed malignancies worldwide. Previous studies have shown that microRNA-449b (miR-449b) functions as a tumor suppressor in many cancers. However, the role of miR-449b in NSCLC is still unknown. In the present study, miR-449b was significantly downregulated in NSCLC samples and cell lines. Bioinformatics analysis revealed that 3'-UTR region of leucine rich repeat containing G protein-coupled receptor 4 (LGR4) mRNA had putative complementary sequences to miR-449b,which was further confirmed by the luciferase assay. Western blotting showed that restoration of miR-449b in NSCLC cells decreased the expression of LGR4. Interestingly, over-expression of miR-449b inhibited growth and invasion of NSCLC cells in vitro. Furthermore, ectopic expression of LGR4 reversed miR-449b-suppressed proliferation and invasion of NSCLC cells. Therefore, the data of the present study demonstrate that miR-449b inhibits tumor cell growth and invasion by targeting LGR4 in NSCLC.

Aberrant Expression of miR-362 Promotes Lung Cancer Metastasis through Downregulation of Sema3A.

miR-362 is a recently discovered member of the microRNA family, and it modulates a variety of physical activities and plays an important role in the occurrence and development of many tumors. However, the biological functions of hsa-miR-362-5p in non-small-cell lung carcinoma (NSCLC) are unknown. Transwell assay and colony formation were used to determine the migration, invasion, and proliferation of NSCLC cells in vitro. A subcutaneous tumor model in nude mice was established to detect NSCLC tumor growth in vivo. The direct binding of miR-362 to the 3′UTR of Semaphorin 3A (Sema3A) was confirmed by luciferase reporter assay. In this study, we found that the level of miR-362 was higher in NSCLC tissues than in adjacent normal tissues and that the level of miR-362 expression was also elevated in five NSCLC cell lines (A549, 95-D, H1299, H292, and H460) relative to a human normal lung epithelial cell line (BEAS2B). Furthermore, miR-362 promoted NSCLC cell invasion, migration, and colony formation in vitro and tumor formation in vivo. Next, we identified the miR-362 target gene Sema3A, which is significantly correlated with metastasis. Sema3A expression was increased in normal tissues relative to NSCLC tissues. This result is consistent with the fact that miR-362 expression is negatively correlated with Sema3A expression in clinical tissue samples and indicated that miR-362 can regulate Sema3A expression in NSCLC cells and consequently affect NSCLC invasion, migration, and colony formation. Taken together, these findings on the newly identified miR-362/Sema3A axis elucidate the molecular mechanism of NSCLC invasion and migration and could lead to a potential therapeutic target in NSCLC treatment.

Abstract 481: QKI, a direct target of miRNA-200, inhibits epithelial-to-mesenchymal transition

Abstract MicroRNA (miR)-200 family members are well-known regulators of epithelial-to-mesenchymal transition (EMT) in epithelial tumors. To identify novel miR-200 targets, we analyzed TargetScan and TCGA data sets. Among 20 candidate genes, we selected QKI gene and studied its role during EMT process in human head and neck squamous cell carcinoma (HNSCC) and non-small cell lung carcinoma (NSCLC) cells. The expression of QKI was proved to negatively correlate with those of miR-200 members in TCGA data sets. miR-200 suppressed QKI expression directly, which was confirmed by 3'-UTR reporter assays. Interestingly, shRNA-mediated knockdown of QKI promoted migration, invasion, and EMT in cancer cells and increased tumor growth in a xenograft mouse model. These results indicate that QKI inhibits EMT and tumorigenesis in HNSCC and NSCLC cells. Even though further studies are necessary to elucidate the underlying mechanism, we suggest that QKI and miR-200 form a feedback loop to maintain homeostatic responses to EMT-inducing signals. Citation Format: Eun Ju KIM, Der Sheng Sun, Hye Sung Won, Yoon Ho Ko, Young-Ho Ahn. QKI, a direct target of miRNA-200, inhibits epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 481.

Abstract 5754: Hypoxia-induced tumor plasticity and immune resistance involves an alteration of target recognition by a mechanism involving TGF-beta signaling

Abstract Backgound: Hypoxia is known to shape the tumor microenvironment of developing tumors and contributes to both tumor progression and escape to immune surveillance. Previous work using non-small cell lung carcinoma cells (NSCLC) showed that hypoxia promotes epithelial-mesenchymal transition (EMT) in a fraction of carcinoma cells and that acquisition of a more mesenchymal (Mes) phenotype by certain cancer subclones correlates with an increased propensity to resist cytotoxic T lymphocytes (CTL) attacks as compared to more Epithelial (Epi) cancer subclones. Moreover, numerous studies identified alterations or loss of major histocompatibility complex (MHC) class I as a potential mechanism of tumor escape in cancer. Here, we asked whether hypoxia-induced EMT could regulate MHC class I expression and whether that could impact on resistance to CTL-mediated killing. Methods and Results : By comparing CMH class I in various NSCLC cancer subclones that have experienced hypoxic stress, we found that cancer clones with enhanced Mes features have a reduced expression of CMH class I molecules, as compared to those with a more Epi phenotype. Transient siRNA knockdown of hypoxia effectors HIF-1α or HIF-2α did not affect the expression of CMH class I molecules suggesting that EMT effectors, rather that HIFs, directly regulate CMH class I expression. Among EMT effectors, TGF-β expression was found to be highly upregulated in Mes cancer clones. Moreover, treatment of these cells with TGF-βR inhibitors markedly upregulated CMH class I expression. and as target cells, increases their susceptibility to CTL-mediated killing. However, blocking of TGF-β using Ab to TGF-β during the coculture time had no significant effects on CTL-mediated killing. This suggests that the observed changes in target cell susceptibility occurs through TGF-β-mediated repression of MHC class I rather than by cancer cell production of TGF-β affecting CTL effector functions. Conclusions: These results suggest that the increase in TGF-β signaling, especially found in EMTed Mes carcinoma cells in response to hypoxic stress, contributes to downregulate CMH class I expression in these cells while coinciding with a reduced susceptibilty to CTL attacks. The development of agents targeting hypoxia or EMT components should provide new therapeutic opportunities in combination with current approaches. Citation Format: Stephane Terry, Stephanie Buart, Jean-Paul Thiery, Fathia Mami-Chouaib, Salem Chouaib. Hypoxia-induced tumor plasticity and immune resistance involves an alteration of target recognition by a mechanism involving TGF-beta signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5754.

Abstract 3813: Development of JNJ-64164711, a low fucose anti-GITR antibody for enhanced depletion of tumor regulatory T cells (Tregs)

Abstract Background: Checkpoint blockade of PD-1/PDL-1 or CTLA-4 has demonstrated clinical benefit in a subset of cancer patients (pts). To further improve the outcome, approaches to deplete immunosuppressive Tregs are being developed. Emerging evidence supports that the TNF receptor superfamily member 18 (TNFRSF18 or GITR) is highly expressed on intratumoral (i.t.) Tregs with limited expression on effector T cells or circulating immune cells (1,2). This differential expression allows for development of monoclonal antibody (mAb) therapies that eliminate this population while sparing other immune subsets. Herein we characterize JNJ-64164711 (JNJ-711), a fully human anti-GITR mAb being developed to selectively deplete GITR+ Tregs and expand the population of pts that may respond to immunotherapy. Methods: Treg frequency and GITR expression on human cancer specimens was performed by IHC and multicolor flow cytometric analysis (FC). JNJ-711 was assessed for binding to GITR (human and cyno) and human Fcγ receptors, and inhibition of hGITRL binding to hGITR. The JNJ-711 binding epitope was mapped by hydrogen-deuterium exchange. JNJ-711 functional activity was determined by 1) an NF-kB reporter assay, 2) antibody dependent cellular cytotoxicity (ADCC) on GITR+ cell lines, and 3) JNJ-711 mediated Treg depletion in primary pts tumor samples. Results: GITR expression prevalence was evaluated by IHC in multiple solid tumors. Immune cell GITR expression was observed in head and neck (HN), esophageal (ESO), non-small cell lung carcinoma (NSCLC), colorectal (CRC) and prostate cancers. GITR was moderately expressed on HN and ESO tumor cells. GITR expression (by FC) was higher on tumor Tregs versus other T cell populations in the tumor and periphery across NSCLC, CRC, ovarian and renal cell carcinoma samples. GITR was differentially higher on i.t. effector Tregs (eTreg) whereas CCR4 was highest on CD4+ T cells in circulation. JNJ-711 binds to hGITR on cells with high affinity [KD= 180 pM], has a log greater affinity to FcγRIIIa [KD= 37 nM on 158V; KD= 180 nM on 158F], a slower dissociation rate than most GITR agonist (Ag) mAb tested [Koff= 5.47 x 10-4 s-1 for Fab], and binds to the CRD1 of GITR. Unlike other GITR Ag mAbs tested, JNJ-711 does not block GITR interaction with GITRL. JNJ-711 demonstrates higher ADCC mediated cell depletion than other GITR Ag mAbs in clinical development on 1) GITR+ hematologic tumor cell lines (HuT102, KMS-12-BM, RPMI 8226, JJN-3), 2) primary activated GITR+ T cells, and 3) in vitro expanded Tregs. Using dissociated primary pts samples, JNJ-711 selectively depleted i.t. eTregs. Conclusion: JNJ-64164711 selectively depletes hGITR+ cells including Tregs and has potential to enhance antitumor immune response. This antibody is being evaluated for clinical development.

Phytochemical naphtho[1,2-b] furan-4,5‑dione induced topoisomerase II-mediated DNA damage response in human non-small-cell lung cancer

BackgroundPhytochemical naphtho[1,2-b] furan-4,5‑dione (NFD) presenting in Avicennia marina exert anti-cancer effects, but little is known regarding about DNA damage-mediated apoptosis in non-small-cell lung carcinoma (NSCLC). PurposeTo examine whether NFD-induced apoptosis of NSCLC cells is correlated with the induction of DNA damage, and to investigate its underlying mechanism. Study designThe anti-proliferative effects of NFD were assessed by MTS Assay Kit FACS assay, and in vivo nude mice xenograft assay. The DNA damage related proteins, the Bcl-2 family and pro-apoptotic factors were examined by immunofluorescence assay, q-PCR, and western blotting. The activity of NF-κB p65 in nuclear extracts was detected using a colorimetric DNA-binding ELISA assay. The inhibitory activity of topoisomerase II (TOPO II) was evaluated by molecular docking and TOPO II catalytic assay. ResultsNFD exerted selective cytotoxicity against NSCLC H1299, H1437 and A549 cells rather than normal lung-embryonated cells MRC-5. Remarkably, we found that NFD activated the hull marker and modulator of DNA damage repairs such as γ-H2AX, ATM, ATR, CHK1, and CHK2 probably caused by the accumulation of intracellular reactive oxygen species (ROS) and inhibition of TOPO II activity. Furthermore, the suppression of transcription factor NF-κB by NFD resulted in significantly decreased levels of pro-survival proteins including Bcl-2 family Bcl-2, Bcl-xL and Mcl-1 and the endogenous inhibitors of apoptosis XIAP and survivin in H1299 cells. Moreover, the nude mice xenograft assay further validated the suppression of H1299 growth by NFD, which is the first report for evaluating the anti-cancer effect of NFD in vivo. ConclusionThese findings provide a novel mechanism indicating the inhibition of TOPO II activity and NF-κB signaling by NFD, leading to DNA damage and apoptosis of NSCLC tumor cells.

Adiponectin inhibits migration and invasion by reversing epithelial‑mesenchymal transition in non‑small cell lung carcinoma.

Adiponectin is the most abundant adipokine in the tumor microenvironment. The role of this protein in tumor progression, however, remains controversial. In the present study, we aimed to investigate the effects of adiponectin on the abilities of migration and invasion in non-small cell lung carcinoma (NSCLC). Using NSCLC cell lines, we examined the effects of adiponectin on cell migration and invasion using Transwell assays. Expression of epithelial-mesenchymal transition markers was examined via microscopy and western blotting. We also performed a knockdown of Twist, AdipoR1 and AdipoR2 in NSCLC cells with siRNAs. The addition of adiponectin to NSCLC cells inhibited both the migration and invasion abilities. Furthermore, we found that NSCLC cells displayed increased epithelial marker expression and downregulation of mesenchymal marker expression following adiponectin administration. Twist AdipoR1 and AdipoR2 knockdown reversed the inhibitory effects of adiponectin on migration and invasion in NSCLC and epithelial-mesenchymal transition. Exogenous adiponectin significantly impaired the migratory and invasive capacities of NSCLC cells through reversal of EMT, suggesting that adiponectin may be a novel promising therapeutic approach against NSCLC.