Metastatic Lung Adenocarcinoma Cell Research Articles

FOXA3 regulates cholesterol metabolism to compensate for low uptake during the progression of lung adenocarcinoma.

Cholesterol metabolism is vital for multiple cancer progression, while how cholesterol affects lung, a low-cholesterol tissue, for cancer metastasis and the underlying mechanism remain unclear. In this study, we found that metastatic lung adenocarcinoma cells acquire cellular dehydrocholesterol and cholesterol by endogenous cholesterol biosynthesis, instead of uptake upon cholesterol treatment. Besides, we demonstrated that exogenous cholesterol functions as signaling molecule to induce FOXA3, a key transcription factor for lipid metabolism via GLI2. Subsequently, ChIP-seq analysis and molecular studies revealed that FOXA3 transcriptionally activated Hmgcs1, an essential enzyme of cholesterol biosynthesis, to induce endogenous dehydrocholesterol and cholesterol level for membrane composition change and cell migration. Conversely, FOXA3 knockdown or knockout blocked cholesterol biosynthesis and lung adenocarcinoma metastasis in mice. In addition, the potent FOXA3 inhibitor magnolol suppressed metastatic gene programs in lung adenocarcinoma patient-derived organoids (PDOs). Altogether, our findings shed light onto unique cholesterol metabolism and FOXA3 contribution to lung adenocarcinoma metastasis.

Establishment of Dual Fluorescent Labeled Human High Bone Metastasis Lung Adenocarcinoma Cell Line and Transcriptomic Characterization Analysis

Bone is a common site for metastasis in lung adenocarcinoma, but the mechanism behind lung adenocarcinoma bone metastasis is still unclear. And currently, there is a lack of easily traceable and stable lung adenocarcinoma bone metastasis cell models, which limits the research on the mechanism of lung adenocarcinoma bone metastasis. The establishment of human lung adenocarcinoma cell line that are highly metastatic to bone, labeled with green fluorescent proteins (GFP) and fireflies luciferase (LUC), along with transcriptomic characterization, would be beneficial for research on lung adenocarcinoma bone metastasis and provide new experimental methods. The human lung adenocarcinoma cell line A549-GFP-LUC was injected into nude mice via the left ventricle to construct a bone metastasis model, and was domesticated in vivo for three consecutive times to obtain the human high bone metastasis lung adenocarcinoma cell line A549-GFP-LUC-BM3; cell counting kit-8 (CCK-8), colony formation assay, scratch wound assays, Transwell assay and Western blot were used to compare the proliferation and invasion abilities of A549-GFP-LUC-BM3 with the parental cells. A549-GFP-LUC-BM3 cells and parental cells were further analyzed by transcriptomic sequencing. Human high-bone metastatic lung adenocarcinoma cells A549-GFP-LUC-BM3 was successfully established. Compared to parental cells, this cells exhibited a significantly higher incidence of bone metastasis and enhanced in vitro proliferation, migration, and invasion abilities. Transcriptomic sequencing results revealed that the A549-GFP-LUC-BM3 cell line had 2954 differentially expressed genes compared to the parental cells, with 1021 genes up-regulated and 1933 genes down-regulated. Gene Ontology (GO) functional enrichment analysis indicated that the differentially expressed genes were primarily localized in cellular components such as the cell periphery. The molecular functions identified as significantly enriched included signaling receptor activity, calcium ion binding, and extracellular matrix structural constituent. Additionally, the biological processes found to be enriched were cell adhesion and biological adhesion. The enrichment analysis conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the differentially expressed genes were primarily involved in the metabolism of xenobiotics by cytochrome P450, retinol metabolism, drug metabolism-cytochrome P450, cell adhesion molecules, steroid hormone biosynthesis, and the nuclear factor kappa B (NF-κB) signaling pathway. The highly bone-metastatic human lung adenocarcinoma cell line with GFP and luciferase double labeling was successfully established. The biological behavior and transcriptome sequencing of the cell line suggest that it has a high bone-metastatic potential.

Single-cell data analysis of malignant epithelial cell heterogeneity in lung adenocarcinoma for patient classification and prognosis prediction

Lung cancer is one of the leading causes of cancer-related death. Most advanced lung adenocarcinoma (LUAD) patients have poor survival because of drug resistance and relapse. Neglecting intratumoral heterogeneity might be one of the reasons for treatment insensitivity, while single-cell RNA sequencing (scRNA-seq) technologies can provide transcriptome information at the single-cell level. Herein, we combined scRNA-seq and bulk RNA-seq data of LUAD and identified a novel cluster of malignant epithelial cells - KRT81+ malignant epithelial cells - associated with worse prognoses. Further analysis revealed that the hypoxia and EMT pathways of these cells were activated to predispose them to differentiate into metastatic lung adenocarcinoma cells. Finally, we also studied the role of these tumor cells in the immune microenvironment and their role in the classification and prognosis prediction of lung adenocarcinoma patients.

Interactive, integrated analysis of single-cell transcriptomic and phylogenetic data with PhyloVision

SummaryRecent advances in CRISPR-Cas9 engineering and single-cell assays have enabled the simultaneous measurement of single-cell transcriptomic and phylogenetic profiles. However, there are few computational tools enabling users to integrate and derive insight from a joint analysis of these two modalities. Here, we describe “PhyloVision”: an open-source software for interactively exploring data from both modalities and for identifying and interpreting heritable gene modules whose concerted expression are associated with phylogenetic relationships. PhyloVision provides a feature-rich, interactive, and shareable web-based report for investigating these modules while also supporting several other data and meta-data exploration capabilities. We demonstrate the utility of PhyloVision using a published dataset of metastatic lung adenocarcinoma cells, whose phylogeny was resolved using a CRISPR-Cas9-based lineage-tracing system. Together, we anticipate that PhyloVision and the methods it implements will be a useful resource for scalable and intuitive data exploration for any assay that simultaneously measures cell state and lineage.

<i>PhyloVision</i>: Interactive Software for Integrated Analysis of Single-Cell Transcriptomic and Phylogenetic Data

Recent advances in CRISPR-Cas9 engineering and single-cell assays have enabled the simultaneous measurement of single-cell transcriptomic and phylogenetic profiles. However, there are few computational tools enabling users to integrate and derive insight from a joint analysis of these two modalities. Here, we describe PhyloVision: an open source software for interactively exploring data from both modalities and for identifying and interpreting heritable gene modules whose concerted expression are associated with phylogenetic relationships. PhyloVisionprovides a feature-rich, interactive, and shareable web-based report for investigating these modules, while also supporting several other data and meta-data exploration capabilities. We demonstrate the utility of PhyloVisionusing a published dataset of metastatic lung adenocarcinoma cells, whose phylogeny was resolved using a CRISPR/Cas9-based lineage-tracing system. Together, we anticipate that PhyloVisionand the methods it implements will be a useful resource for scalable and intuitive data exploration for any assay that simultaneously measures cell state and lineage.

Upregulation of CD109 Promotes the Epithelial-to-Mesenchymal Transition and Stemness Properties of Lung Adenocarcinomas via Activation of the Hippo-YAP Signaling.

Metastasis is the leading cause of death in lung adenocarcinomas. Identifying potential prognostic biomarkers and exploiting regulatory mechanisms could improve the diagnosis and treatment of lung cancer patients. We previously found that cluster of differentiation 109 (CD109) was upregulated in lung tumor tissues, and CD109 overexpression was correlated with the invasive and metastatic capacities of lung adenocarcinoma cells. However, the contribution of CD109 to lung tumorigenesis remains to be elucidated. In the present study, we identified that CD109 was upregulated in metastatic lung adenocarcinoma cells, and elevation of CD109 was correlated with epithelial-to-mesenchymal transition (EMT) traits in patients with lung adenocarcinoma. Functionally, CD109 expression was crucial for EMT gene expressions, tumor invasiveness, and cancer stemness properties. Moreover, elevation of CD109 was accompanied by upregulation of the yes-associated protein (YAP) signature in metastatic lung cancer cells and lung cancer patients, and activation of YAP was demonstrated to participate in CD109-elicited EMT gene expressions and tumor invasiveness. Our study reveals the molecular mechanism underlying CD109 in lung tumor aggressiveness, and CD109 could be a potential diagnostic and therapeutic target for lung cancer patients.

Abstract 1841: Adaptive upregulation of carbamoyl phosphate synthetase-1 (CPS-1) in glucose-deprived metastatic lung adenocarcinoma cells

Abstract Advanced lung cancers frequently metastasize to the brain, where nutrient deprivation from tumour microenvironments at either primary, invading or metastatic sites may exert a selective pressure on tumour cells. Our lab has identified a rare brain metastasis cell line of lung cancer origin: A1115, that possessed heightened basal glycolytic activity. Characterization of the metabolic profiles of A1115 and 4 other lung adenocarcinoma cell lines revealed A1115 to have the highest dependence on glycolysis and the lowest utilization of mitochondrial oxidative phosphorylation (OXPHOS) for metabolism. Thus, we hypothesized that A1115 is hypersensitive to glucose starvation. A1115 relied heavily on glycolysis on survival and propagation, while inhibiting mitochondrial OXPHOS had less effect. Glucose deprivation induced the nutrient sensor AMP-activated protein kinase (AMPK) to be elevated in A1115, while the proliferative capacity of A1115 was drastically reduced by an AMPK activator, AICAR, suggesting AMPK to be detrimental to cell viability of A1115 under glucose deprivation. To understand how such metastatic cell line could survive the low glucose environment in blood circulation and during the invasion into the brain parenchyma, we established clonal A1115 sublines that could propagate under low glucose (LG) environment. LG sublines regained hypersensitivity to glucose deprivation upon exposure to normal glucose concentrations suggest an adaptive response. We hypothesised that tumour cells maintain cell viability under glycolytic stress conditions by undergoing metabolic reprogramming, this involves changes in metabolite utilization and transcriptional activity of key metabolic enzymes. A parallel metabolomics (targeted LC/MS) and transcriptomics (RNAseq) analysis was conducted comparing A1115 LG sublines with the parental control. Metabolomics analysis revealed that LG sublines under glucose deprivation harboured a significant metabolite increase in amino acids: glutamate, arginine, aspartic acid and pyrimidine: cytidine, whereas integrated pathway analysis of metabolomics and transcriptomics revealed upregulation in both pyrimidine metabolism and alanine, aspartate and glutamate metabolism. Moreover, increased carbamoyl phosphate synthetase 1 (CPS-1) expression was observed in LG sublines, an initiating enzyme for pyrimidine biosynthesis and urea cycle, silencing CPS-1 decreased cell viability on LG in normal conditioned media, suggesting that the disruption of pathway could impede survival. Overall, our results suggest that lung adenocarcinoma may undergo reprogramming towards a starkly different constituent of transcriptional activation leading to metabolite flux and signalling activation to allow viable adaption to glucose deprivation in the microenvironment. Citation Format: Kin Lok Wong, Andrew M. Chan. Adaptive upregulation of carbamoyl phosphate synthetase-1 (CPS-1) in glucose-deprived metastatic lung adenocarcinoma cells [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 1841.

Spatially controlled photo-patterning of multi-material bioactive hydrogels

Event Abstract Back to Event Spatially controlled photo-patterning of multi-material bioactive hydrogels Bagrat Grigoryan1*, Jonathon D. Roybal2, Paul T. Greenfield1, Alexander J. Zaita1, Samantha J. Paulsen1, Jacob L. Albritton1, Anderson Ta1, Don L. Gibbons2, 3 and Jordan S. Miller1 1 Rice University, Bioengineering, United States 2 The University of Texas M.D. Anderson Cancer Center, Thoracic/Head and Neck Medical Oncology, United States 3 The University of Texas, MD Anderson Cancer Center, Molecular and Cellular Oncology, United States Introduction: While fabrication of simple microenvironments to study cell fate and function has been achieved using soft lithography and photolithography techniques, mimicking the complex, heterogeneous patterns of native tissue in vitro remains challenging due to lack of available simple, automated patterning technologies[1]. To this end, we developed a stereolithography (SLA)-based 3D printer for automated fabrication of hydrogels containing microstructures composed of multi-materials for applications towards understanding the lung tumor microenvironment. Materials and Methods: We developed and characterized our custom SLA-based 3D printer to spatially control the patterning of multiple materials within a single layer and across successive layers. We performed photorheological characterization of prepolymer formulations containing poly(ethylene glycol) diacrylate (PEGDA) and lithium acylphosphinate (LAP) photoinitiator to quantitatively understand the gelation kinetics and modulus of a single layer of hydrogel. Patterned entrapped fluorophore molecules facilitated characterization of the fidelity of multi-material patterns. To demonstrate biocompatibility of our technology, we added hMSCs into the prepolymer solution and performed LIVE/DEAD staining of printed cell-laden hydrogels at various time points. We further demonstrate the biocompatibility of our technology by adding a murine metastatic lung adenocarcinoma cell line, 344SQ, into the prepolymer solution to fabricate bioactive PEG-based gels containing cell-adhesive CGRGDS ligand and MMP-sensitive peptides incorporated into the polymer backbone[2]. We investigated cell viability and function by monitoring the innate ability of 344SQ cells to form into aggregates and lumenize over time in the proper microenvironment. Results and Discussion: The photorheological assay provided us with information on the exposure duration needed for a certain layer thickness as a function of PEG molecular weight, PEG concentration, and LAP concentration in the prepolymer formulation for reproducible fabrication of hydrogels (Figure 1A). Fluorescent microscopy images demonstrated fabrication of features as small as 300µm in diameter as well as precise spatial control in photo-patterning of multiple materials within the same plane (Figure 1B). Encapsulation of cells in bioactive PEG-based gels demonstrated cell viability greater than 90% after 2 days of culture (Figure 1C-Top). Encapsulated 344SQ cells retained their biological activity by forming into aggregates and lumenizing throughout the 2-week long culture (Figure 1C-Bottom). Conclusion: We have developed an open-source SLA-based 3D printer that enables us to automatically fabricate hydrogels containing bioactive microstructures with spatial control of multiple materials at a medium-high throughput. We utilized this technology to encapsulate hMSCs and metastatic cells in PEG-based hydrogels with high cell viability and function. We believe the automated nature of our developed system will enable researchers to recapitulate intricate microenvironments for studying complex cellular interactions in vitro without facing difficulties associated with precisely aligning masks with traditional photolithographic techniques. Thanks to the Open Source Hardware Association, RepRap.org, and related projects and companies that support worldwide, open standardization of 3D printing. This work was supported in part by NSF Graduate Research Fellowship (BG).

Overexpression of Long Non-Coding RNA ZXF2 Promotes Lung Adenocarcinoma Progression Through c-Myc Pathway

Objective: To investigate the expression of long non-coding RNA ZXF2 in lung adenocarcinoma tissues and its effect on cell proliferation, migration and invasion. Methods: Forty pairs of cancerous and adjacent non-cancerous lung adenocarcinoma specimens were collected for the studies. Quantitative real-time PCR was used to analyze the expression of ZXF2 in tumor tissues and adjacent normal tissues. The expression of ZXF2 was correlated with patients' clinico-pathological data. Molecular pathway controlled by ZXF2 was explored by using small interfering RNA (siRNA) technology. CCK-8 cell proliferation assay, flow cytometry analysis and transwell assays were used to evaluate cell proliferation, migration and invasion. Results: The expression of ZXF2 was 2 fold or higher in 27 out of 40 (67.5%) cases of lung adenocarcinoma specimens than that in non-cancerous tissues (P<0.05). The relative expression level of ZXF2 was positively correlated with tumor lymph node metastasis (χ²=8.485, P<0.05) and poor prognosis of the patients (p=0.0217). In order to explore the molecular mechanisms of ZXF2 mediated tumor progression, ZXF2 expression was inhibited by siRNA in A549 cells, a highly aggressive and metastatic lung adenocarcinoma cell line. We found that siRNA-ZXF2 treatment inhibited cell proliferation (P<0.01) leading to cell cycle arrest (P<0.01). The cell migration and invasion were suppressed by siRNA-ZXF2 treatment (P<0.01). Further biochemical studies revealed that the knockdown of ZXF2 led to down regulation of c-Myc signaling. Conclusion: ZXF2 was overexpressed in lung adenocarcinoma tissues and the high expression of ZXF was closely related to tumor progression through c-Myc related pathway. Given the fact that both ZXF2 and c-Myc are located in the same chromosome 8q24.2 loci, the potential interaction between ZXF2 and c-Myc might be a novel target for treatment of lung adenocarcinoma.

S100B promotes the proliferation, migration and invasion of specific brain metastatic lung adenocarcinoma cell line

Brain metastasis frequently occurs in cancer patients and is associated with a poor prognosis. We previously reported that S100B was highly expressed in PC14/B, a specific brain metastatic lung adenocarcinoma cell line, which suggests that it is associated with brain metastasis of lung cancer. However, the role of S100B in brain metastasis remains to be elucidated. In this study, using PC14/B cell line, we found that siRNA mediated depletion of S100B in PC14/B cells led to notable differences in cell proliferation, apoptosis, cell cycle progression, colony formation ability, cell migratory and invasive activity compared with the mock-transfected cells. Therefore, our data suggest that S100B promotes the brain metastasis of lung adenocarcinoma by promoting cell proliferation, preventing apoptosis and increasing cell migration and invasion.

TSG101, identified by screening a cancer cDNA library and soft agar assay, promotes cell proliferation in human lung cancer

Understanding the genesis and development of tumors is an essential component in cancer research. It is of interest to discover unknown genes that are responsible for cellular transformation. A cDNA library of a highly metastatic lung adenocarcinoma cell line was constructed. This library was introduced into the NIH3T3 mouse embryonic fibroblast cell line to screen for cDNAs that increase anchorage-independent colony formation in soft agar. The expression of TSG101 in lung cancer cell lines and specimens was confirmed using reverse transcription-polymerase chain reaction. The level of TSG101 protein in transfected A549 cells was determined by western blotting. Cell-cycle distribution was analyzed using a FACStar Plus flow cytometer. One of the candidate cDNAs that increases anchorage-independent colony formation was shown to correspond to the TSG101 cDNA sequence. Levels of TSG101 mRNA were higher in lung cancer cell lines and specimens compared to matched normal lung tissues. Ectopic expression of TSG101 in the A549 lung adenocarcinoma cell line increased the numbers of cells in S phase, suggesting an increased cell proliferation rate. These results indicate that TSG101 may induce the malignant phenotype of cells.

Analysis of UPA induced proteolytic activity in extracellular matrix of human highly metastatic lung adenocarcinoma cell line

Analysis of UPA induced proteolytic activity in extracellular matrix of human highly metastatic lung adenocarcinoma cell line