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Nitrilase 1 modulates lung tumor progression in vitro and in vivo.

ABSTRACT: Uncovering novel growth modulators for non-small cell lung cancer (NSCLC) may lead to new therapies for these patients. Previous studies suggest Nit1 suppresses chemically induced carcinogenesis of the foregut in a mouse model. In this study we aimed to determine the role of Nit1 in a transgenic mouse lung cancer model driven by a G12D Kras mutation. Nit1 knockout mice (Nit1-/-) were crossed with KrasG12D/+ mice to investigate whether a G12D Kras mutation and Nit1 inactivation interact to promote or inhibit the development of NSCLC. We found that lung tumorigenesis was suppressed in the Nit1-null background (Nit1-/-:KrasG12D/+). Micro-CT scans and gross tumor measurements demonstrated a 5-fold reduction in total tumor volumes compared to Nit1+/+KrasG12D/+ (p<0.01). Furthermore, we found that Nit1 is highly expressed in human lung cancer tissues and cell lines and use of siRNA against Nit1 decreased overall cell survival of lung cancer cells in culture. In addition, cisplatin response was enhanced in human lung cancer cells when Nit1 was knocked down and Nit1-/-:KrasG12D/+ tumors showed increased sensitivity to cisplatin in vivo. Together, our data indicate that Nit1 may play a supportive role in the modulation of lung tumorigenesis and represent a novel target for NSCLCs treatment.

SUBMITTER: Wang YA

PROVIDER: S-EPMC5008292 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Nitrilase 1 modulates lung tumor progression in vitro and in vivo.

Wang Yong Antican YA Sun Yunguang Y Le Blanc Justin M JM Solomides Charalambos C Zhan Tingting T Lu Bo B

Oncotarget 20160401 16

Uncovering novel growth modulators for non-small cell lung cancer (NSCLC) may lead to new therapies for these patients. Previous studies suggest Nit1 suppresses chemically induced carcinogenesis of the foregut in a mouse model. In this study we aimed to determine the role of Nit1 in a transgenic mouse lung cancer model driven by a G12D Kras mutation. Nit1 knockout mice (Nit1-/-) were crossed with KrasG12D/+ mice to investigate whether a G12D Kras mutation and Nit1 inactivation interact to promot ...[more]

PMID: 26967383

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Project description:Three-dimensional (3D) cell culture is gaining acceptance in response to the need for cellular models that better mimic physiologic tissues. Spheroids are one such 3D model where clusters of cells will undergo self-assembly to form viable, 3D tumor-like structures. However, to date little is known about how spheroid biology compares to that of the more traditional and widely utilized 2D monolayer cultures. Therefore, the goal of this study was to characterize the phenotypic and functional differences between lung tumor cells grown as 2D monolayer cultures, versus cells grown as 3D spheroids. Eight lung tumor cell lines, displaying varying levels of epidermal growth factor receptor (EGFR) and cMET protein expression, were used to develop a 3D spheroid cell culture model using low attachment U-bottom plates. The 3D spheroids were compared with cells grown in monolayer for 1) EGFR and cMET receptor expression, as determined by flow cytometry, 2) EGFR and cMET phosphorylation by MSD assay, and 3) cell proliferation in response to epidermal growth factor (EGF) and hepatocyte growth factor (HGF). In addition, drug responsiveness to EGFR and cMET inhibitors (Erlotinib, Crizotinib, Cetuximab [Erbitux] and Onartuzumab [MetMab]) was evaluated by measuring the extent of cell proliferation and migration. Data showed that EGFR and cMET expression is reduced at day four of untreated spheroid culture compared to monolayer. Basal phosphorylation of EGFR and cMET was higher in spheroids compared to monolayer cultures. Spheroids showed reduced EGFR and cMET phosphorylation when stimulated with ligand compared to 2D cultures. Spheroids showed an altered cell proliferation response to HGF, as well as to EGFR and cMET inhibitors, compared to monolayer cultures. Finally, spheroid cultures showed exceptional utility in a cell migration assay. Overall, the 3D spheroid culture changed the cellular response to drugs and growth factors and may more accurately mimic the natural tumor microenvironment.

Project description:ObjectiveAberrant epigenetic remodeling represents a molecular hallmark in lung adenocarcinoma (LUAD). We aim to investigate the biological roles of SETDB2 and its underlying associations with oxidative stress, providing therapeutic targets for individualized treatment of LUAD.MethodsDifferential analysis was conducted via Limma package, and Kaplan-Meier analysis was performed with survival package. CCK-8, cell proliferation assay, transwell assay, and in vivo assays were conducted to assess the function of SETDB2. Western blot assay, RT-qPCR, and immunohistochemistry (IHC) were conducted to assess the expression levels of SETDB2/NRF2. Chromatin immunoprecipitation (ChIP) assay and ChIP-qPCR were conducted to assess the epigenetic roles of SETDB2.ResultsWe found that SETDB2 expression is decreased in tumor samples versus normal tissues in TCGA-LUAD cohort, LUAD-EAS cohort, GSE72094 dataset, and independent Soochow-LUAD dataset. Patients with low SETDB2 levels had a worse prognosis relative to those with high SETDB2. SETDB2 inhibition could significantly promote cell growth, migration ability, and stemness maintenance. Gene set enrichment analysis (GSEA) suggested that SETDB2 correlated with oxidative stress crosstalk and regulated NRF2 mRNA levels. ChIP assay suggested that SETDB2 mainly recruited the H3K9me3 enrichment at the NRF2 promoter region to suppress the mRNA levels of NRF2. Downregulated SETDB2 could activate NRF2 transcription and expression, thereby promoting its downstream targets, like NQO1, FTH1, and ME1. Functional experiments demonstrated that low SETDB2 allowed NRF2 to drive malignant processes of LUAD. SETDB2 overexpression attenuated the ability of NRF2 signaling to neutralize cellular reactive oxygen species (ROS) levels, leading to enhanced cell apoptosis. Overexpressed SETDB2 could inhibit tumor progression in vivo and further render LUAD cells sensitive to chemotherapy.ConclusionsIn conclusion, these findings uncovered the suppressive role of SETDB2 in LUAD. SETDB2 negatively regulates NRF2 signaling to modulate tumor progression, which creates a therapeutic vulnerability in LUAD.

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Nitrilase 1 modulates lung tumor progression in vitro and in vivo.

Publications

Nitrilase 1 modulates lung tumor progression in vitro and in vivo.

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