Lung Squamous Carcinoma Cell Line Research Articles

S100A6 could not promote the differentiation of Calu-6 lung cancer cell line.

Our previous study demonstrated that S100 calcium binding protein A6 (S100A6) impairs tumorigenesis by Calu-6 lung cancer cells, as well as inhibit their growth. However, the role that S100A6 plays in tumor cell differentiation has not been previously explored. This study aimed to confirm the effect of S100A6 on the direction of differentiation in the human lung cancer cell linem Calu-6m based on our previous published research. A S100A6-overexpressing lentiviral vector was successfully constructed in our previous study. Nude mouse tumorigenicity was then applied successfully, and 15 mice were divided into three groups (Calu-6, Calu-6/neo, Calu-6/S100A6). After 5 weeks, we detected lung cancer markers with immunohistochemistry in mice tumor tissues, including the adenocarcinoma markers, TTF-1 and NapsinA, the squamous cell carcinoma markers, P40, CK5/6 and P63, and the small cell lung cancer markers CD56, Syn, CgA, TTF-1, CK, and Ki-67. Differences among the three groups were statistically compared. All the above-mentioned markers were positive in the tumor tissues of all three groups, and there were no significant differences. S100A6 cannot promote differentiation of the undifferentiated human lung cancer cell line, Calu-6, into adenocarcinoma, squamous, or small cell carcinoma cell lines.

Expression and prognosis analysis of TBX2 subfamily in human lung carcinoma.

Lung cancer has a high morbidity and mortality rate of all cancers worldwide. Therefore, there is an urgent need for reliable cancer markers for diagnosis and prognosis of patients with lung cancer. In this study, we used the bioinformatics database to compare the expression of the TBX2 subfamily at the transcriptional and protein levels in non-small cell lung cancer. Then, to confirm our bioinformatics analysis above, we used western bloting to determine the expression of TBX2, TBX3, TBX4 and TBX5 in human lung squamous carcinoma cell lines. Besides, low expression of TBX2 subfamily predicted a poor prognosis of patients with lung cancer. Finally, The methylation database was used to explore the relationship between the low expression of TBX2 subfamily and methylation of gene promoter region. Our data showed a significant decrease of TBX2 subfamily expression in lung cancer tissues of several histological subtypes. Finally, the methylation of TBX2 subfamily members in the promoter region of NSCLC was significantly higher than that in normal tissues. Our research provided sufficient evidence that TBX2 subfamily might play an inhibitory role in malignancy progression of lung cancer, which is promising to shed light on discovering a novel reliable cancer marker for prognosis of lung cancer patients.

ANKRD49 promotes the metastasis of NSCLC via activating JNK-ATF2/c-Jun-MMP-2/9 axis

BackgroundAnkyrin repeat domain 49 (ANKRD49) has been found to be highly expressed in multiple cancer including lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC). However, the function of ANKRD49 in the pathogenesis of NSCLC still remains elusive. Previously, ANKRD49 has been demonstrated to promote the invasion and metastasis of A549 cells, a LUAD cell line, via activating the p38-ATF-2-MMP2/MMP9 pathways. Considering the heterogeneity of tumor cells, the function and mechanism of ANKRD49 in NSCLC need more NSCLC-originated cells to clarify.MethodsReal-time qPCR was employed to test ANKRD49 expression levels in nine pairs of fresh NSCLC tissues and the corresponding adjacent normal tissues. The function of ANKRD49 was investigated using overexpression and RNA interference assays in lung adenocarcinoma cell line (NCI-H1299) and lung squamous carcinoma cell line (NCI-H1703) through gelatin zymography, cell counting kit-8, colony formation, wound healing, migration and invasion assays mmunoprecipitation was performed to in vitro. Immunoprecipitation was performed to test the interaction of c-Jun and ATF2. Chromatin immunoprecipitation was conducted to assess the transcriptional regulation of ATF2/c-Jun on MMP-2/9. Moreover, the tumorigenicity of ANKRD49 was evaluated in nude mice models and the involved signal molecular was also measured by immunohistochemical method.ResultsWe found that the levels of ANKRD49 in cancerous tissues were higher than those in adjacent normal tissues. in vitro assay showed that ANKRD49 promoted the migration and invasion of NCI-H1299 and NCI-H1703 cells via enhancing the levels of MMP-2 and MMP-9. Furthermore, ANKRD49 elevated phosphorylation of JNK and then activated c-Jun and ATF2 which interact in nucleus to promote the binding of ATF2:c-Jun with the promoter MMP-2 or MMP-9. In vivo assay showed that ANKRD49 promoted lung metastasis of injected-NSCLC cells and the high metastatic rate was positively correlated with the high expression of ANKRD49, MMP-2, MMP-9, p-JNK, p-c-Jun and p-ATF2.ConclusionThe present study indicated that ANKRD49 accelerated the invasion and metastasis of NSCLC cells via JNK-mediated transcription activation of c-Jun and ATF2 which regulated the expression of MMP-2/MMP-9. The molecular mechanisms of ANKRD49’s function is different from those found in A549 cells. The current study is a supplement and improvement to the previous research.

MiR-101-3p targets KPNA2 to inhibit the progression of lung squamous cell carcinoma cell lines.

We herein discuss the impacts of miR-101-3p on the tumorigenesis-related cell behaviors in lung squamous cell carcinoma (LUSC) by repressing KPNA2. TCGA database was utilized to measure miR-101-3p and KPNA2 levels in LUSC tissues and cells. The interaction of miR-101-3p and KPNA2-3'UTR was determined by dual luciferase assay. Western blot evaluated the protein level of KPNA2. MiR-101-3p was under-expressed in LUSC cells while KPNA2 was overexpressed. Western blot confirmed the impact of KPNA2 expression on cancer cell progression. The negative regulatory impact of miR-101-3p on KPNA2 was also verified. In vitro cell function assays revealed the suppressing effect of high miR-101-3p expression on cell invasion, migration and viability, as well as its promoting effect on apoptosis. Up-regulated miR-101-3p weakened the promoting effect of overexpressed KPNA2 on LUSC malignant progression. To conclude, miR-101-3p repressed viability, invasion, and migration, and facilitated cell apoptosis in LUSC by suppressing KPNA2.

Characterization and mechanisms of radioresistant lung squamous cell carcinoma cell lines.

Radiotherapy is an important clinical treatment for patients with lung squamous cell carcinoma (LUSC), and resistance to radiotherapy is an important cause of recurrence and metastasis in LUSC. The aim of this study was to establish and explore the biological characteristics of radioresistant LUSC cells. The LUSC cell lines NCI-H2170 and NCI-H520 were irradiated (4 Gy × 15Fraction). Radiosensitivity, cell apoptosis, cell cycle, and DNA damage repair were measured by clonogenic survival assay, flow cytometry, immunofluorescence for γ-H2AX foci, and Comet assay, respectively. Activation of p-ATM(Ser1981), p-CHK2(Th68), p-DNA-PKcs (Ser2056), and Ku70/Ku80 was measured by western blot. Proteomics was used to explore the differential genes and enriched signaling pathways between radioresistant cell lines and parental lines. In vivo nude mouse xenograft experiments further verified the feasibility of the radioresistant LUSC cell lines. After fractionated irradiation (total dose of 60 Gy), radioresistant cells had decreased radiosensitivity, increased G0/G1 phase arrest, enhanced DNA damage repair ability, and through the ATM/CHK2 and DNA-PKcs/Ku70 pathways regulated double strands break. The upregulated differential genes in radioresistant cell lines were mainly enriched in biological pathways such as cell migration and extracellular matrix (ECM)-receptor interaction. In vivo verification of decreased radiosensitivity of radioresistant cells CONCLUSIONS: Radioresistant LUSC cell lines were established by fractional radiotherapy, which regulates IR-induced DNA damage repair through ATM/CHK2 and DNA-PKcs/Ku70. Tandem Mass Tags (TMT) quantitative proteomics found that the biological process pathway of cell migration and ECM-receptor interaction are upregulated in LUSC radioresistant cells.

Abstract 2392: Multi-dimensional morphology analysis enables identification and label-free enrichment of heterogeneous tumor cell populations

Abstract Studying tumor heterogeneity at single cell resolution is crucial for elucidating tumor progression and treatment response. Classically, tumor cells are characterized with a limited set of biomarkers that cannot cover the full extent of the tumor cell heterogeneity. The advent of single cell genomics has enabled scientists to study heterogeneity at high resolution, but gene expression signatures may not translate to readily available biomarkers for functional studies. Multi-dimensional morphology analysis has the potential for higher resolution than cell surface biomarkers while also reducing cell manipulations. The Deepcell platform enables multi-dimensional morphology analysis of unlabeled single cells using artificial intelligence (AI), imaging, and microfluidics, allowing for higher resolution of population heterogeneity beyond protein expression markers. Using the Deepcell platform, we trained an AI model to identify and sort for malignant cells from non-small cell lung cancer cells (NSCLC) tumor biopsies based on morphology. scRNA-seq, CNV, and targeted mutation analysis verified enrichment of carcinoma cells. Sorted cells had gene expression signatures indicative of tumor cells (e.g. EpCAM expression), increased genomic alterations by CNV analysis, and increased allele frequency of P53 and KRAS mutations relative to pre-sorted samples. Overall, the data indicate brightfield images of cells can be used to detect macro-level changes in cell morphology resulting from the molecular events involved in tumorigenesis. In addition, we induced chemotherapeutic drug resistance of lung cancer cell lines in vitro, imaged the resulting cultures on the Deepcell platform, and used the bright-field cell images to generate multi-dimensional morphological embeddings that can be visualized by UMAP. The resulting UMAPs showed distinct clusters of cells for both the parent and resistant cell lines, indicating that there are morphological differences associated with drug resistance. We developed a random forest classifier to identify the top differential morphological features between parental and resistant cell lines. These top features can distinguish between the populations with high accuracy (87%). Finally, we performed multi-dimensional morphology analysis to compare lung adenocarcinoma (LA) and squamous cell carcinoma (SCC) cell lines. The classifier predicted the correct cell type with 75% accuracy, suggesting that differences between these two carcinomas are reflected in their morphology. Together, our data suggest that AI-detected morphological differences between cell populations may show a biologically significant link between morphology and phenotype. As such, multi-dimensional morphology analysis will bolster the understanding of complex disease states and tumor microenvironment, particularly in patient derived biopsies. Citation Format: Andreja Jovic, Kiran Saini, Ryan Carelli, Tiffine Pham, Christian Corona, Jeanette Mei, Michael Phelan, Stephane C. Boutet, Kevn Jacobs, Julie Kim, Manisha Ray, Chassidy Johnson, Nianzhen Li, Mahyar Salek, Maddison Masaeli, Matt Barnes, Cyril Ramathal. Multi-dimensional morphology analysis enables identification and label-free enrichment of heterogeneous tumor cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2392.

MP20-07 NPTX1 IS A NOVEL SERUM BIOMARKER INDUCED BY ARPI TREATED PROSTATE CANCER AND ENRICHED IN LINEAGE PLASTICITY WITH BRN2 HIGH

You have accessJournal of UrologyCME1 Apr 2023MP20-07 NPTX1 IS A NOVEL SERUM BIOMARKER INDUCED BY ARPI TREATED PROSTATE CANCER AND ENRICHED IN LINEAGE PLASTICITY WITH BRN2 HIGH Takeshi Namekawa, Joshua Scurll, Maxim Kobelev, Dwaipayan Ganguli, Olena Sivak, Martin Gleave, Vancouver, Canada, Eva Corey, Colm Morrissey, and Amina Zoubeidi Takeshi NamekawaTakeshi Namekawa More articles by this author , Joshua ScurllJoshua Scurll More articles by this author , Maxim KobelevMaxim Kobelev More articles by this author , Dwaipayan GanguliDwaipayan Ganguli More articles by this author , Olena SivakOlena Sivak More articles by this author , Martin GleaveMartin Gleave More articles by this author , Vancouver Vancouver More articles by this author , Canada Canada More articles by this author , Eva CoreyEva Corey More articles by this author , Colm MorrisseyColm Morrissey More articles by this author , and Amina ZoubeidiAmina Zoubeidi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Androgen receptor (AR) pathway inhibition is the first-line therapy for metastatic prostate cancer (PCa). However, tumors invariably develop resistance to ARPI. We previously identified a transcription factor Brain-2 (BRN2), encoded by the gene POU3F2, as a master driver of NEPC which is an aggressive form of AR-independent phenotypes treatment resistant PCa. In this study, we identified a surrogate biomarker of BRN2 activity to monitor the progression of treatment resistant PCa. METHODS: To identify BRN2 serum marker, we analyzed BRN2 ChIPseq data from 2 NEPC and 2 neural-type lung squamous cell carcinoma (nLUSC) cell lines with a focus on shared BRN2 peaks. Annotated peaks from enhancers (EnhancerAtlas) and promoters (genes within 20kb) were selected for secreted proteins and were ranked by correlation with BRN2 expression in PCa patients. Validation of protein expression was performed by western blot. R-PLEX Assay kit was used to measure concentrations in culture media from NCI-H660 cells and in serum from mice with NCI-H660 xenograft or PDX tumors and from human PCa patients. RESULTS: We identified 51 candidate serum biomarkers for BRN2 activity (or expression?) using the methods described above. These biomarkers were ranked based on their expression and correlation with BRN2 expression in PCa patient samples across four public data sets. NPTX1 was identified as the top biomarker of BRN2 expression. We further validated association of NPTX1 and BRN2 expression by BRN2 overexpression in MR42D and 22Rv1, indicating that BRN2 positively regulates NPTX1 expression. Next, we showed that NPTX1 is present in culture media from NCI-H660 cells, and that NPTX1 serum concentration positively correlated with tumor volume in mice with NCI-H660 xenografts and with NPTX1 mRNA expression in LuCaP PDX models. Importantly, evaluation of patients’ samples showed that NPTX1 levels were high in 26/35 CRPC serum samples vs levels in serum of healthy volunteers, and that the NPTX1 serum levels were highest in 4/6 patients with high POU3F2 tumor expression. CONCLUSIONS: We identified and validated NPTX1 as a serum marker that can be readily detected in serum from NEPC patients with BRN2high tumours. Source of Funding: U.S. Army Medical Research and Development Command(W81XWH1810689) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e277 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takeshi Namekawa More articles by this author Joshua Scurll More articles by this author Maxim Kobelev More articles by this author Dwaipayan Ganguli More articles by this author Olena Sivak More articles by this author Martin Gleave More articles by this author Vancouver More articles by this author Canada More articles by this author Eva Corey More articles by this author Colm Morrissey More articles by this author Amina Zoubeidi More articles by this author Expand All Advertisement PDF downloadLoading ...

An efficient synthesis of O- and N- alkyl derivatives of 4-aminobenzoic acid and evaluation of their anticancer properties

A series of twenty alkyl derivatives (2–21) of 4-amino benzoic acid (1, PABA) have been prepared using potassium carbonate and opportune alkylating agents under simple and mild reaction conditions. Compounds (16–21) are reported for the first time. Electron impact mass spectrometry (EIMS), Fourier transform infrared (FTIR) and Proton nuclear magnetic resonance (1H-NMR) spectroscopic techniques were adopted for the characterization of these analogues. In the present study, the cytotoxic screening of sixteen compounds (3, 5–11, 13 and 15–21) was also achieved against lung (NCI-H460) and oral squamous carcinoma (CAL-27) cell lines. Compound 20 has shown magnificent inhibitory properties against NCI-H460 cell line (IC50 15.59 and 20.04 µM, respectively) at a lower dose than that of the control (cisplatin; IC50 21.00 µM). One-way analysis of variance (ANOVA), t-test and Pearson correlation coefficient (PCC) have been performed to determine the reliability of current data through statistical package for the social sciences (SPSS).

Discovery of EP300/CBP histone acetyltransferase inhibitors through scaffold hopping of 1,4-oxazepane ring

EP300 and its paralog CBP play an important role in post-translational modification as histone acetyltransferases (HATs). EP300/CBP inhibition has been gaining attention as an anticancer treatment target in recent years. Herein, we describe the identification of a novel, highly selective EP300/CBP inhibitor, compound 11 (DS17701585), by scaffold hopping and structure-based optimization of a high-throughput screening hit 1. Compound 11 (DS17701585) shows dose-dependent inhibition of SRY-box transcription factor 2 (SOX2) mRNA expression in a human lung squamous cell carcinoma cell line LK2-xenografted mouse model.

MiR-139-3p Targets CHEK1 Modulating DNA Repair and Cell Viability in Lung Squamous Carcinoma Cells.

Non-small-cell lung carcinoma (NSCLC) can be classified into several subtypes, where lung squamous carcinoma (LUSC) is one common subtype. Though miR-139-3p has been reported to be implicated in the development of various cancers, its mechanisms and functions remain unclear in LUSC. In this study, miR-139-3p was screened as one of the significantly down-regulated miRNAs in LUSC by an "edgeR" differential analysis based on TCGA database, which was verified by qRT-PCR in LUSC cell lines as well. The viability and cell cycle of the LUSC cells were examined by CCK-8 and flow cytometry, respectively, exhibiting that upregulating miR-139-3p restrained cell viability and thus accelerating the cell cycle. To explain this phenomenon, we further explored the downstream target gene through miRTarBase and starBase databases, where CHEK1 was predicted as one candidate. The targeting relationship was verified by a dual luciferase assay, identifying that CHEK1 could be targeted by miR-139-3p. Then, qRT-PCR and western blot analyses were performed to detect the expression of CHEK1 mRNA and proteins under the alteration of miR-139-3p expression. Rescue experiments were conducted to confirm the impacts of miR-139-3p/CHEK1 axis on the cell viability and cell cycle of LUSC. The results indicated that the effects of miR-139-3p on the LUSC cell phenotypes could be blocked by overexpressing CHEK1. In conclusion, our study provides a novel insight into the regulatory role of miR-139-3p in the development of LUSC.

MiR-30a-5p suppresses lung squamous cell carcinoma via ATG5 - mediated autophagy.

Propose: Autophagy plays a complicated role in cancer progression. This study aims at assessing the function of ATG5-induced autophagy in progression of lung squamous cell carcinoma and its upstream mechanism.Method: TCGA database of lung squamous cell carcinoma was analyzed to explore the differentially expressed miRNAs and mRNAs and relative prognosis. RT-PCR and Western blot were performed to evaluate autophagy relative gene expression level in human lung squamous cell carcinoma cell Lines. Autophagy flux was observed using transmission electron microscopy and immunofluorescence. Meanwhile, binding relationship of potential target miRNA and mRNAs were also confirmed using Dual-luciferase reporter gene assay. Lung metastatic model was established to evaluated the effect of targeting protein and miRNA.Result: High level expression of ATG5 was detected in LUSC patients. Relative experiments confirmed that ATG5 silencing could decrease the autophagy flux in LUSC. In addition, our research revealed that there is a binding sites between hsa-mir-30a-5p and 3′-UTR of ATG5. Mimic miR-30a-5p suppresses ATG5-mediated autophagy in lung squamous cell carcinoma cells. The in vivo experiments confirmed that miR-30a-5p could attenuate lung squamous cell carcinoma progression through the autophagy pathway.Conclusion: Accordingly, the in vivo and in vitro study in our research have demonstrated that miR-30a-5p inhibits lung squamous cell carcinoma progression via ATG5-mediated autophagy.

Overexpression of MicroRNA-340-5p Attenuates Lung Cancer Cell Proliferation and Invasion through Wnt/ Beta-Catenin Signaling Pathway

Numerous studies have screened and dysregulated the microRNA in various cancers via microarray assay. In this dysregulated microRNA, microRNA-340-5p attracted our attention due to its high conservation and fold change. MicroRNA-340-5p is abnormally expressed in several cancers and plays an important role. However, the expression and function of microRNA-340-5p in lung cancer are still unknown. Our aim was to explore the role of microRNA-340-5p in lung cancer and its underlying molecular mechanism. The lung cancer cell lines of human lung squamous cell carcinoma cell line SKMES1, human lung adenocarcinoma cell line A549, lung adenocarcinoma cells H23, human lung cancer cell line SPC-A1 and human squamous cell carcinoma H520 were used in present study. Real time polymerase chain reaction was performed to detect microRNA-340-5p and beta-catenin messenger RNA expression. Transwell assay was conducted to determine the invasion of human squamous cell carcinoma H520 cells under different treatment conditions. Our study showed that the expression of microRNA-340-5p in lung cancer tissues and was significantly lower than normal tissues. Moreover, overexpression of microRNA-340-5p could suppress the invasion and proliferation of lung cancer cells. We also confirmed that microRNA-340-5p and beta-catenin are negatively correlated in lung cancer tissues and the role of microRNA-340-5p in lung cancer can be reversed by overexpression of beta-catenin. Our results demonstrate that up-regulation of microRNA-340-5p attenuates lung cancer cell invasion and proliferation via inhibiting wingless-related integration site/beta-catenin pathway. The data will provide new insights for the treatment of lung cancer and new guidance for clinical treatment of lung cancer.

Investigation of Targeting Relationship between Micro-Rna-22 and Vegfr3 in Lung Squamous Cell Carcinoma.

The present study explored the clinical significance of microRNA-22 (miR-22) expression in lung squamous cell carcinoma and to explore the targeting relationship with vascular endothelial growth factor receptor 3 (VEGFR3). A total of 49 patients with lung squamous cell carcinoma who underwent surgical treatment were selected. The expression of miR-22 was detected by fluorescence quantitative realtime PCR (qPCR), the expression of VEGFR3 was detected by Western blotting assays, and D240 labeled microlymphatic vessels density (MLVD) was detected by immunohistochemistry (IHC). Lung squamous cell carcinoma cell line SK-MES-1 was selected and the targeting relationship between miR-22 and VEGFR3 was analyzed by double luciferase reporter gene assay. Western blotting assays were used to detect the expression of vascular endothelial growth factor-D (VEGFD) and D240 in the blank control group, empty vector transfection group, miR-22 transfection group, miR-22 and VEGFR3 co-transfection group. The expression range of miR-22 in lung squamous cell carcinoma was 0.8-3.5. The expression of miR-22 in lung squamous cell carcinoma was significantly different by tumor maximum diameter, lymph node metastasis, vascular invasion and TNM stage. The expression of miR-22 was linked to survival time. There was a negative correlation between miR-22 and VEGFR3, miR-22 and MLVD. Double luciferase reporter gene assays showed that miR-22 reduced the luciferase activity of pGL3-VEGFR3-WT transfected cells. Compared with the control group, the expression of VEGF-D and D2-40 in the miR-22 transfection group was significantly decreased. However, VEGF-D and D240 in the miR-22 and VEGFR3 co-transfection group reversed the changes. We assumed that the abnormal expression of miR-22 in lung squamous cell carcinoma may be involved in the development and progression of lung squamous cell carcinoma. MiR-22 negatively regulated the target gene VEGFR3 to mediate lymphangiogenesis. The expression of miR-22 may also be linked to the prognosis of the disease.

The novel proautophagy anticancer drug ABTL0812 potentiates chemotherapy in adenocarcinoma and squamous nonsmall cell lung cancer.

Around 40% of newly diagnosed lung cancer patients are Stage IV, where the improvement of survival and reduction of disease-related adverse events is the main goal for oncologists. In this scenario, we present preclinical evidence supporting the use of ABTL0812 in combination with chemotherapy for treating advanced and metastatic Nonsmall cell lung adenocarcinomas (NSCLC) and squamous carcinomas. ABTL0812 is a new chemical entity, currently in Phase 1b/2a clinical trial for advanced squamous NSCLC in combination with paclitaxel and carboplatin (P/C), after successfully completing the first-in-human trial where it showed an excellent safety profile and signs of efficacy. We show here that ABTL0812 inhibits Akt/mTOR axis by inducing the overexpression of TRIB3 and activating autophagy in lung squamous carcinoma cell lines. Furthermore, treatment with ABTL0812 also induces AMPK activation and ROS accumulation. Moreover, combination of ABTL0812 with chemotherapy markedly increases the therapeutic effect of chemotherapy without increasing toxicity. We further show that combination of ABTL0812 and chemotherapy induces nonapoptotic cell death mediated by TRIB3 activation and autophagy induction. We also present preliminary clinical data indicating that TRIB3 could serve as a potential novel pharmacodynamic biomarker to monitor ABTL0812 activity administered alone or in combination with chemotherapy in squamous NSCLC patients. The safety profile of ABTL0812 and its good synergy with chemotherapy potentiate the therapeutic potential of current lines of treatment based on chemotherapy regimens, arising as a promising option for improving these patients therapeutic expectancy.

Mechanism of lung adenocarcinoma spine metastasis induced by CXCL17.

Spine metastases are common in patients with lung adenocarcinoma (LUAD). Improving the clinical outcome of spine metastasis LUAD patients requires knowledge on the mechanism underlying the metastatic process. Here, we sought to decipher the effect and mechanism of C-X-C motif chemokine ligand 17 (CXCL17) on LUAD spine metastasis. Clinical tumor tissue samples, lung cancer cell lines and a TCGA dataset were used for CXCL17 expression analyses. A transwell invasion assay was used to assess the chemotaxis capacity of mononuclear macrophages induced by CXCL17. Western blotting was performed to explore the mechanism of mononuclear macrophage chemotaxis towards CXCL17. A cell counting kit-8 assay was employed to investigate the effect of conditioned medium from M1 and M2 macrophages on the proliferation of lung cancer cells. We found that the expression of CXCL17 was higher in clinical LUAD samples and LUAD cell lines than in lung squamous cell carcinoma (LUSC) samples and cell lines. Moreover, we found that CXCL17 increased the migration of THP-1 mononuclear macrophages by activating the Src/FAK pathway. In addition, we found that conditioned medium from M2 macrophages promoted the proliferation of LUAD cells. From our data we conclude that CXCL17 is a key regulator of LUAD spine metastasis. CXCL17 and its downstream Src/FAK pathway may serve as clinical intervention targets.

Abstract 2983: LpMab-23: an anti-podoplanin cancer-specific antibody for a prognostic marker of oral cancer

Abstract Background Podoplanin (PDPN) is a specific ligand of C-type lectin-like recepotor-2 (CLEC-2), and is involved in cancer metastasis. The physiological PDPN function has been discussed in many normal tissues, including lymphatic endothelial cells, pulmonary type I alveolar cells, and renal podocytes. Herein, we aimed to produce cancer-specific anti-PDPN monoclonal antibodies (mAbs). Furthermore, we investigated whether the reactivity of cancer-specific anti-PDPN mAbs might be a prognostic marker of oral cancer. Materials and methods We immunized mice with LN229/PDPN cells, and screened mAbs, which react with PDPN-expressing cancer cell lines, such as glioblastoma cell lines and lung squamous cell carcinoma cell lines and do not react with PDPN-expressing normal cells, including primary lymphatic endothelial cells and renal epithelial cell lines in flow cytometry. Furthermore, we investigated the reactivity of anti-PDPN mAbs by immunohistochemistry against oral cancers. Anti-tumor activities by mouse-human chimeric LpMab-23 (chLpMab-23) were examined using mouse xenograft models of oral cancer cells. Finally, we analyzed the association between the reactivity of anti-PDPN mAbs and clinical/pathological features of oral cancers. Results We established LpMab-23, a cancer-specific anti-PDPN mAb. LpMab-23 reacted with PDPN-expressing cancer cell lines in flow cytometry. In contrast, LpMab-23 recognized PDPN-expressing normal cells very weakly. LpMab-23 reacted only with PDPN-expressing cancer cells, not with lymphatic endothelial cells in oral cancer tissues by immunohistochemistry. The epitope mapping of anti-PDPN mAbs revealed that LpMab-23 recognized a cancer-specific glycopeptide including Thr55/Ser56. chLpMab-23 revealed high ADCC and anti-tumor activities against oral cancers. The Kaplan-Meier curves of the five-year new metastasis-free survival rate (nMFS) were significantly lower in LpMab-23-positive patients than in the patients with LpMab-23-negative ones. Conclusions A cancer-specific anti-PDPN mAb (LpMab-23) and its mouse-human chimeric mAb (chLpMab-23) were successfully established. LpMab-23 is advantageous in anti-tumor activities. LpMab-23-positive cases could be a useful predictor of poor prognosis for oral cancer. Citation Format: Takashi Miwa, Shinji Yamada, Mika K. Kaneko, Yoshikazu Furusawa, Masato Fukui, Yukinari Kato. LpMab-23: an anti-podoplanin cancer-specific antibody for a prognostic marker of oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2983.

Pterostilbene inhibits lung squamous cell carcinoma growth in vitro and in vivo by inducing S phase arrest and apoptosis.

Natural dietary components have become the subject of an increasing amount of interest due to the side effects of anticancer treatment. Pterostilbene, an analog of resveratrol, is primarily found in grapes, and has been suggested to exert antioxidant and anticancer effects in different tumor types. The present study aimed to investigate the antitumor effects and molecular mechanisms of pterostilbene in the human lung squamous cell carcinoma (SqCC) cell line, H520. The results of the present study indicate that pterostilbene significantly reduced cell viability and induced S phase arrest, and that treatment with pterostilbene was associated with the downregulation of cyclin A and cyclin E, as with the upregulation of p21 and p27 expression in H520 cells. In the apoptosis analysis, pterostilbene induced S phase accumulation and the activation of caspase-3, −8 and −9 in H520 cells, potentially through the activation of extrinsic and intrinsic apoptotic pathways. Additionally, the in vivo study demonstrated that pterostilbene effectively inhibited lung SqCC growth in a H520 ×enograft model. Given the in vitro and in vivo antitumor effects of pterostilbene demonstrated in the present study, pterostilbene may serve a novel and effective therapeutic agent to for patients with SqCC.

Pathways regulating the expression of the immunomodulatory protein glycodelin in non‑small cell lung cancer.

Glycodelin [gene name, progesterone associated endometrial protein (PAEP)] was initially described as an immune system modulator in reproduction. Today, it is also known to be expressed in several types of cancer, including non-small cell lung cancer (NSCLC). In this cancer type, the feasibility of its usage as a follow-up biomarker and its potential role as an immune system modulator were described. It is assumed that NSCLC tumours secrete glycodelin to overcome immune surveillance. Therefore, targeting glycodelin might be a future approach with which to weaken the immune system defence of NSCLC tumours. In this context, it is important to understand the regulatory pathways of PAEP/glycodelin expression, as these are mostly unknown so far. In this study, we analysed the influence of several inducers and of their downstream pathways on PAEP/glycodelin expression in a human lung adenocarcinoma carcinoma (ADC; H1975) and a human lung squamous cell carcinoma (SQCC) cell line (2106T). PAEP/glycodelin expression was notably stimulated by the canonical transforming growth factor (TGF)-β pathway in SQCC cells and the PKC signalling cascade in both cell lines. The PI3K/AKT pathway inhibited PAEP/glycodelin expression in the ADC cells and an antagonizing role towards the other investigated signalling cascades is suggested herein. Furthermore, the mitogen-activated protein kinase kinase (MEK)/extracellular-signal regulated kinases (ERK) pathway was, to a lesser extent, found to be associated with increased PAEP/glycodelin amounts. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), MEK/ERK pathway and TGF-β are targets of NSCLC drugs that are already approved or are currently under investigation. On the whole, the findings of this study provide evidence that inhibiting these targets affects the expression of glycodelin and its immunosuppressive effect in NSCLC tumours. Moreover, understanding the regulation of glycodelin expression may lead to the development of novel therapeutic approaches with which to weaken the immune system defence of NSCLC tumours in the future.

Effects of lemur tyrosine kinase-3 on cell proliferation and invasion in lung squamous carcinoma cell line SK-MES-1

Effects of lemur tyrosine kinase-3 on cell proliferation and invasion in lung squamous carcinoma cell line SK-MES-1

Abstract B22: Exposure of AZD4547 FGFR-TKI to a lung squamous carcinoma cell line induces resistance by small cell lung cancer-like transformation associated with ASCL1

Abstract Molecular targeted medicine has progressed in non-small cell lung cancer in recent years, and acquired resistance to molecular targeted therapy has become a major problem. Several mechanisms of acquired resistance have been described, such as secondary mutation of targeted genes, bypass pathway activation, and phenotypic transformation, including epithelial-mesenchymal transition and small cell lung cancer transformation. However, detailed mechanisms and the strategy to overcome the resistance have yet to be clarified thoroughly. In this study, we established a squamous lung cancer cell line with acquired resistance to FGFR tyrosine kinase inhibitor in vitro and identified achaete-scute family bHLH transcription factor 1 (ASCL1) overexpression as a mechanism of the resistance to tyrosine kinase inhibitor treatment. We identified LK-2 as a squamous cell lung cancer cell line that is highly sensitive to FGFR tyrosine kinase inhibitor AZD4547, which is currently evaluated in clinical trial in squamous cell lung cancer, and established its resistant clone in vitro by exposing gradually increasing concentration of AZD4547. The resistant clone was morphologically more round-shaped and expressed ASCL1 significantly higher than the original LK-2 in a microarray analysis, but did not have additional mutations in putative genes responsible for resistance in RNA-sequencing. ASCL1 inhibition with shRNA lentivirus did not restore the sensitivity to AZD4547, but significantly suppressed the growth of the resistant clone compared to the original LK-2. Cyclin A1 (CCNA1) expression was also increased in the resistant clone and was regulated by ASCL1. CCNA1 inhibition also suppressed the growth of the resistant clone. These results suggest that ASCL1 was one of the key molecules for the acquired resistance to tyrosine kinase inhibitor therapy, and considering the evidence that ASCL1 is a master regulator of small cell lung cancer-like phenotype, it may be related to small cell lung cancer transformation as a mechanism of the resistance. ASCL1 and its downstream molecule CCNA1 may be candidates to overcome the resistance to tyrosine kinase inhibitor therapy. Citation Format: Yosuke Amano, Takahiro Ando, Keita Maemura, Toshio Sakatini, Mitsuhiro Sunohara, Kosuke Watanabe, Hidenori Kage. Exposure of AZD4547 FGFR-TKI to a lung squamous carcinoma cell line induces resistance by small cell lung cancer-like transformation associated with ASCL1 [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B22.