Proliferation Of H1299 Cells Research Articles

Tumor Necrosis Factor α–Dependent Lung Inflammation Promotes the Progression of Lung Adenocarcinoma Originating From Alveolar Type II Cells by Upregulating MIF-CD74

Lung adenocarcinoma is the most common type of lung cancer. We recently reported that inflammation-driven lung adenocarcinoma (IDLA) originates from alveolar type (AT)-II cells, which depend on major histocompatibility complex (MHC) class II to promote the expansion of regulatory T cells. The MHC class II–associated invariant chain (CD74) binds to the macrophage migration inhibitory factor (MIF), which is associated with promoting tumor growth and invasion. However, the role of MIF-CD74 in the progression of lung adenocarcinoma and the underlying mechanisms remain unclear. We aimed to explore the role of MIF-CD74 in the progression of lung adenocarcinoma and elucidate the mechanisms by which tumor necrosis (TNF)-α–mediated inflammation regulates CD74 and MIF expression in IDLA. In human lung adenocarcinoma, CD74 was upregulated on the surface of tumor cells originating from AT-II cells, which correlated positively with lymph node metastasis, tumor origin/nodal involvement/metastasis stage, and TNF-α expression. MIF interaction with CD74 promoted the proliferation and migration of A549 and H1299 cells in vitro. Using a urethane-induced IDLA mouse model, we observed that CD74 was upregulated in tumor cells and macrophages. MIF expression was upregulated in macrophages in IDLA. Blocking TNF-α–dependent inflammation downregulated CD74 expression in tumor cells and CD74 and MIF expression in macrophages in IDLA. Conditioned medium from A549 cells or activated mouse AT-II cells upregulated MIF in macrophages by secreting TNF-α. TNF-α–dependent lung inflammation contributes to the progression of lung adenocarcinoma by upregulating CD74 and MIF expression, and AT-II cells upregulate MIF expression in macrophages by secreting TNF-α. This study provides novel insights into the function of CD74 in the progression of IDLA.

Effects of Cinobufagin on the Proliferation, Migration, and Invasion of H1299 Lung Cancer Cells.

Cinobufagin (CB), with its steroidal nucleus structure, is one of the major, biologically active components of Chan Su. Recent studies have shown that CB exerts inhibitory effects against numerous cancer cells. However, the effects of CB regarding the metastasis of non-small cell lung cancer (NSCLC) and the involved mechanisms need to be further studied. The purpose of the present study aimed to report the inhibitory function of CB against proliferation and metastasis of H1299 cells. CB inhibited proliferation of H1299 lung cancer cells with an IC50 value of 0.035±0.008 μM according to the results of MTT assays. Antiproliferative activity was also observed in colony forming cell assays. In addition, 5-ethynyl-2'-deoxyuridine (EdU) retention assays revealed that CB significantly inhibited the rate of DNA synthesis in H1299 cells. Moreover, results of the scratch wound healing assays and transwell migration assays displayed that CB exhibited significant inhibition against migration and invasion of H1299 cells. Furthermore, CB could concentration-dependently reduce the expression of integrin α2, β-catenin, FAK, Src, c-Myc, and STAT3 in H1299 cells. These western blotting results indicated that CB might target integrin α2, β-catenin, FAK and Src to suppress invasion and migration of NSCLC, which was consistent with the network pharmacology analysis results. Collectively, findings of the current study suggest that CB possesses promising activity against NSCLC growth and metastasis.

LIM-domain binding protein 2 was down-regulated by miRNA-96-5p inhibited the proliferation, invasion and metastasis of lung cancer H1299 cells

ObjectivesLung cancer was one of the most common malignancies around the world. It has great significance in to search for the mechanism of occurrence and development of lung cancer. LIM Domain Binding protein 2 (LDB2) belongs to the LIM-domain binding family, it can be used as a binding protein that combined with other transcription factors to form the transcription complex for regulating the expression of target genes. The expression of microRNA-96-5p (miR-96-5p) has been investigated in various tumors. The aim of this study is to investigate the potential role of LDB2 and miR-96-5p in lung cancer. MethodsReal-time quantitative PCR was applied to detect the expression of LDB2 and miR-96-5p. The proliferation, invasion, and metastasis of H1299 cells were analyzed by CCK8, transwell, and wound healing assay after LDB2 or miR-96-5p transfection. Luciferase activities assay and western blot were used to reveal the targeted regulation between LDB2 and miR-96-5p. ResultsHere the authors found LDB2 was down-regulated in lung cancer tissues and negatively correlated with miR-96-5p expression, it could promote or inhibit the proliferation, invasion and metastasis of H1299 cells after LDB2 knockdown or overexpression and regulate the expression of cyclinD1, MMP9, Bcl-2, and Bax via ERK1/2 signaling pathway. Furthermore, miR-96-5p exerted its function by directly binding to 3′-UTR of LDB2 and regulating expression of LDB2. miR-96-5p could promote the proliferation, invasion, and metastasis of H1299 cells. ConclusionThese findings demonstrate that LDB2 can act as a new regulator to inhibit cell proliferation, invasion, and metastasis via the ERK1/2 signaling pathway, and miR-96-5p may be a potential promising molecular by targeting LDB2 in lung cancer.

Structural Characterization of an Alcohol-Soluble Polysaccharide from Bletilla striata and Antitumor Activities in Vivo and in Vitro.

In general, Bletilla striata polysaccharides were mostly water-soluble. However, the structural property, immunomodulatory effects and antitumor activities of alcohol-soluble Bletilla striata polysaccharide were rarely reported. In this study, an alcohol-soluble Bletilla striata polysaccharide was firstly extracted, investigated the structural property and evaluated the antitumor activity in vivo and in vitro. Results showed that BSAP was a low molecular weight polysaccharide (2.29×104 Da) and consisted of glucose, xylose and mannose (molar ratio: 2.39 : 1.00 : 0.21). Animal experiments results suggested that BSAP could effectively inhibit the expansion of H22 solid tumors, protect thymus and spleen, improve macrophages, lymphocytes and NK cells activities and enhance lymphocyte subsets proportion, presenting a better immunological enhancement effect in vivo. Additionally, the results of cell experiments showed that BSAP had obvious antitumor effect in vitro, including inhibiting the proliferation of H22 cells and inducing the apoptosis of tumor cells. These results would provide theoretical basis and new ideas for the further development and utilization of BSAP in the biomedical field.

IL-17A Promotes the Migration, Invasion and the EMT Process of Lung Cancer Accompanied by NLRP3 Activation.

Lung cancer is a deadly cancer worldwide, and its pathogenesis and treatment methods require continuous research and exploration. As a representative factor of adaptive immunity, the role of interleukin-17A (IL-17A) in lung cancer is still unclear. The purpose of the present study was to investigate the effect of IL-17A on the biological behaviour of lung cancer cells and the relative mechanism. The human lung adenocarcinoma A549 and H1299 cell lines were used for in vitro study. The effects of IL-17A on cell proliferation, migration and invasion were assessed by CCK-8 assay, wound-healing assay, transwell invasion assay and real-time cell analysis (RTCA). The expression levels of marker proteins in the process of epithelial-mesenchymal transition (EMT) were detected by western blot analysis. Caspase-1 activity and the concentration of IL-1β after NLRP3 inflammasome activation were measured by a Caspase-1 Activity Assay Kit and an IL-1β ELISA kit, respectively. Compared to the control group, IL-17A treatment did not affect the proliferation of A549 and H1299 cells in vitro, but it promoted cell migration, invasion and the EMT process. IL-17A treatment increased NLRP3 expression, caspase-1 activity and IL-1β level. Blockade of NLRP3 alleviated the cell migration, invasion and the EMT process induced by IL-17A. In conclusion, these findings indicated that NLRP3 participates in the migration, invasion and the EMT process of IL-17A-stimulated lung cells in vitro.

DDX17 promotes the growth and metastasis of lung adenocarcinoma

DEAD box RNA helicase 17 (DDX17) has been shown to be an RNA binding protein involved in RNA metabolism and associated with cancer progression. However, the biological role of DDX17 in the pathogenesis of lung adenocarcinoma (LUAD) has not been well characterized. Here, we demonstrated that DDX17 promoted the proliferation, migration and invasion of H1299 and A549 lung adenocarcinoma cells. Analyses of public datasets showed that DDX17 is upregulated in LUAD specimens. Our tumor xenograft models confirmed the in vivo promoting role of DDX17 in the growth and metastasis of LUAD. Mechanistic analyses further revealed that DDX17 protein interacts with the mRNA of MYL9 and MAGEA6 and upregulates their levels. MYL9 could mediate the function of DDX17 to regulate the actin cytoskeleton rearrangement and cell adhesion, particularly by modulating the stress fiber and focal adhesion formation, whereas DDX17 might inhibit the autophagy process through MAGEA6/AMPKα1 axis in LUAD cells. Collectively, our study revealed the oncogenic role and pathways of DDX17 in LUAD.

Penthorum chinense Pursh polysaccharide induces a mitochondrial-dependent apoptosis of H22 cells and activation of immunoregulation in H22 tumor-bearing mice

Penthorum chinense Pursh has been widely used as a Chinese traditional functional food for liver disease prevention and therapy. A novel cold-water soluble polysaccharide (PCP-4) has been prepared from Penthorum chinense Pursh at 4 °C. PCP-4 contained both α and β configurations and mainly consisted of galactose, arabinose, galacturonic acid, rhamnose, and glucose (molar ratio: 6.38:5.26:1.69:1:0.87) with an average molecular weight of 1.83 × 106 Da. In vitro experiments results demonstrated that PCP-4 effectively inhibits the proliferation of H22 cells and blocks cells at S phase in a dose-dependent manner. Otherwise, PCP-4-treated cells exhibited typical morphological features of apoptotic cells. Combined the results from western blot analysis, Annexin V-FITC/PI staining, Rhodamine 123 staining and DCFH-DA staining, PCP-4 was found to induce H22 cell apoptosis in vitro via the mitochondrial signaling pathway. The results of in vivo experiments suggested that PCP-4 significantly suppresses xenograft tumor growth by protecting immune organs, enhancing immune cells functionality, and inducing apoptosis. Overall, these results clearly show that Penthorum chinense Pursh polysaccharide serves as a valuable natural product for hepatocellular carcinoma therapy.

M2 macrophages-derived exosomal miR-3917 promotes the progression of lung cancer via targeting GRK6.

Macrophages in the tumor microenvironment (TME) can serve as potential targets for therapeutic intervention. The aim of this study was to investigate the molecular mechanism by which M2 macrophage-derived exosomes (M2-Ex) affect lung cancer progression through miRNA transport. The THP-1 cells were differentiated into M0 and M2 macrophages. M2-Ex were isolated and identified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Cancer tissues and adjacent tissues of non-small-cell lung cancer (NSCLC) patients were collected. H1299 and A549 cells were co-cultured with M2-Ex. Subcutaneous xenograft mouse model was established. miR-3917 is highly expressed in lung cancer tissues and M2-Ex. Interference of miR-3917 in M2-Ex inhibits H1299 cell proliferation, migration and invasion, while overexpression of miR-3917 had the opposite effect in A549 cells. M2-Ex promote tumor growth by delivering miR-3917 in vivo. miR-3917 could target G protein-coupled receptor kinase 6 (GRK6), and interference of miR-3917 in M2-Ex inhibits H1299 cells proliferation, migration and invasion by up-regulating GRK6 level, while overexpression of miR-3917 had the opposite effect in A549 cells. M2-Ex can transfer miR-3917 into lung cancer cells and promote lung cancer progression, providing theoretical basis for the diagnosis and effective treatment of lung cancer.

Design, synthesis, and biological evaluation of 2-arylamino-4-(piperidin-4-yloxy)pyrimidines as potent EGFRT790M/L858R inhibitors to treat non-small cell lung cancer

Three types of 2-arylamino-4-(piperidin-4-yloxy)pyrimidines (I–III) were designed and synthesized as covalent EGFR(epidermal growth factor receptor)T790M/L858R inhibitors by replacement of the common reported 4-(3-amino)phenoxyl moiety with 4-(4-hydroxy)piperidine-4-oxyl, and the introduction of fused-thiophene or -pyrrole on the pyrimidine core to strategically achieve conformational restriction. According to our biological evaluation, it was found that compound 9i could potently suppress EGFRT790M/L858R kinase and H1975 cell proliferation, with IC50 values of 4.902 nM and 0.6210 μM, respectively. Further study showed that 9i not only demonstrated highly selective inhibitory effects toward EGFRT790M/L858R over wild-type EGFR (EGFRWT), but it also had low cytotoxicity against normal HBE(human bronchial epithelial) and L-02 cells. Action mechanism studies showed that 9i effectively hindered cell migration and promoted apoptosis by AO(Acridine Orange)/EB(Ethidium Bromide) staining. These data would provide important clues for the screening of novel covalent EGFRT790M/L858R inhibitors for non-small cell lung cancer (NSCLC) treatment.

Identification and Exploration of Serine Peptidase Inhibitor Kazal Type I (SPINK1) as a Potential Biomarker Correlated with the Progression of Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Although significant advances have been achieved in the treatment of NSCLC during the past two decades, the 5-year survival rate of patients with NSCLC remains <20%. Thus, there is an urgent requirement to further understand the molecular mechanisms that promote NSCLC development and to identify novel therapeutic targets. In the present study, the gene expression profiles of patients with NSCLC from The Cancer Genome Atlas database were carefully analyzed and SPINK1 was identified as a tumor-inducing factor. SPINK1 expression level was found to be increased in both NSCLC tissues and cell lines. Moreover, SPINK1 promoted cell proliferation in A549 and H1299 cells. Knockdown of SPINK1 could activate cell autophagy and apoptosis. Mechanistically, SPINK1 was demonstrated to induce the proliferation of NSCLC via activating the MEK/ERK signaling pathway. In conclusion, these findings suggested that SPINK1 may serve as a potential biomarker in NSCLC.

Mechanism of Puerariae Lobatae Radix against lung cancer by inhibiting histone demethylase LSD1

Histone lysine-specific demethylase 1(LSD1) has become a promising molecular target for lung cancer therapy. Upon the screening platform for LSD1 activity, some Chinese herbal extracts were screened for LSD1 activity inhibition, and the underlying mechanism was preliminarily investigated at both molecular and cellular levels. The results of LSD1 inhibition showed that Puerariae Lobatae Radix extract can effectively reduce LSD1 expression to elevate the expression of H3 K4 me2 and H3 K9 me2 substrates in H1975 and H1299 cells. Furthermore, Puerariae Lobatae Radix was evaluated for its anti-lung cancer activity. It had a potent inhibitory ability against the proliferation and colony formation of both H1975 and H1299 cells. Flow cytometry and DAPI staining assays indicated that Puerariae Lobatae Radix can induce the apoptosis of lung cancer cells. In addition, it can significantly suppress the migration and reverse the epithelial-mesenchymal transition(EMT) process of lung cancer cells by activating E-cadherin and suppressing the expression of N-cadherin, slug and vimentin. To sum up, Puerariae Lobatae Radix displayed a robust inhibitory activity against lung cancer, and the mechanism may be related to the down-regulation of LSD1 expression to induce the cell apoptosis and suppress the cell migration and EMT process. These findings will provide new insights into the action of Puerariae Lobatae Radix as an anti-lung cancer agent and offer new ideas for the study on the anti-cancer action of Chinese medicine based on the epigenetic modification.

Transcription factor AP2 enhances malignancy of non-small cell lung cancer through upregulation of USP22 gene expression

BackgroundUbiquitin-specific protease 22 (USP22), a putative cancer stem cell marker, is frequently upregulated in cancers, and USP22 overexpression is associated with aggressive growth, metastasis, and therapy resistance in various human cancers including lung cancer. However, USP22 gene amplification seldom occurs, and the mechanism underlying USP22 upregulation in human cancers remains largely unknown.MethodsA luciferase reporter driven by a promoter region of USP22 gene was selectively constructed to screen against a customized siRNA library targeting 89 selected transcription factors to identify potential transcription factors (TFs) that regulate USP22 expression in human non-small cell lung cancers (NSCLC). Association of identified TFs with USP22 and potential role of the TFs were validated and explored in NSCLC by biological assays and immunohistochemistry analysis.ResultsLuciferase reporter assays revealed that SP1 and activating transcription factor 3 (ATF3) inhibit USP22 transcription, while transcription factor AP-2 Alpha/Beta (TFAP2A/2B) and c-Myc promote USP22 transcription. Binding site-directed mutagenesis and chromosome immunoprecipitation (ChIP) assays validated AP2α and AP2β are novel TFs of USP22. Furthermore, overexpression of AP2A and AP2B significantly upregulates USP22 expression, and its target: Cyclin D1, concurrently enhances the proliferation, migration, and invasion of NSCLC A549 and H1299 cells in a partially USP22-dependent manner. Moreover, AP2 protein level correlated with USP22 protein in human NSCLC tissues.ConclusionOur findings indicate AP2α and AP2β are important transcription factors driving USP22 gene expression to promote the progression of NSCLC, and further support USP22 as a potential biomarker and therapeutic target for lung cancer.1HTL99SXtPtQRdWEzUs9UTVideo

Inhibitory effects of total triterpenoids isolated from the Hedyotis diffusa willd on H1975 cells

It is estimated that non-small-cell lung cancer (NSCLC) is responsible for 80% of human deaths related to lung cancer worldwide. Currently, it has been discovered that two transcription factors. Nuclear factor-κB (NF-κB) and Signal transducer and activator of transcription 3 (STAT3) were the main factors affecting inflammation and cancer, and their activation promoted lung cancer cell proliferation. Hedyotis diffusa Willd. (H. diffusa) is an herbal Chinese medicine, which has always been used for the treatment of malignant tumors in clinical. Previous research found that H. diffusa could inhibit the proliferation of H1975 cells, but the specific mechanisms remain elusive. We investigated the effects of total triterpenes extracted from H. diffusa (TTH) on the migration, proliferation and apoptosis of H1975 cells. Cell-cycle and immunofluorescence analysis showed that TTH could block H1975 cells at G0/G1 phase and induce apoptosis of experimental cells. The protein levels of Bcl-2 were decreased, while the levels of pro-apoptotic Bax were increased. In addition, TTH could also inhibit the migration of H1975 cells through downregulated MMP-2 and MMP-9 and upregulated TIMP-2. Further research found that the level of phospho-STAT3 was significantly decreased after administration of TTH. And protein expression level of NF-κB in nucleus was decreased after TTH treatment, while NF-κB in cytoplasm increased. These results suggested that TTH could inhibit the proliferation and migration of H1975 cells, and also could induce cell apoptosis. These effects were closely connected to the activation of NF-κB and the phosphorylation of STAT3.

CMaf-Inducing Protein Promotes LUAD Proliferation and Metastasis by Activating the MAPK/ERK Pathway.

Objective Previous studies have shown that cMaf-inducing protein (CMIP) promotes tumorigenesis and progression, however, the role of CMIP in lung adenocarcinoma (LUAD) and its molecular mechanism remain unclear. Methods In this study, the Human Protein Atlas and Kaplan–Meier Plotter database were used to analyze the expression and prognostic value of CMIP in LUAD. Then, the expression levels of CMIP in LUAD tissues and cells were detected by qRT-PCR and western blot. The lentiviral vector was used to establish a stable transfected cell line, and the transfection efficiency was detected by qRT-PCR. MTT assay, colony formation assay, transwell assay, and wound healing assay were used to evaluate the function of CMIP in LUAD. In addition, the effect of CMIP on the MAPK/ERK pathway in LUAD cells was analyzed by western blot. Results The expression level of CMIP was significantly increased in LUAD cell and tissue samples, and the high expression of CMIP was associated with overall survival (OS) and progression-free survival (PFS) in LUAD patients. In vitro experiments showed that CMIP overexpression significantly promoted the proliferation, migration, and invasion of A549 cells. CMIP knockout significantly inhibited the proliferation, migration, and invasion of H1299 cells. In addition, it was observed that the expression levels of the MAPK/ERK pathway-related proteins were significantly increased in CMIP-overexpressed A549 cells, and promoted cell proliferation, migration, and invasion, while U0126 could significantly reverse the activation of the MAPK/ERK pathway by CMIP overexpression, and inhibit the proliferation, migration, and invasion of A549 cells. Conclusion Our study shows that CMIP, as an oncogene, is associated with poor patient prognosis, and may promote the proliferation and metastasis of LUAD by activating the MAPK/ERK pathway. Therefore, CMIP may be a new potential therapeutic target for LUAD.

Long non-coding RNA BZRAP1-AS1 functions in malignancy and prognosis for non-small-cell lung cancer.

PurposeThe function of BZRAP1-AS1 is unknown in lung cancer. We evaluated the clinicopathologic significance of BZRAP1-AS1, and its role in non-small-cell lung cancer (NSCLC) progression.Patient and methodsSixty-three NSCLC patients from Beijing Chest Hospital were included. The expression of BZRAP1-AS1 was detected by real-time quantitative polymerase chain reaction (RT-qPCR) in tumor tissues and adjacent normal tissues. Then, the clinicopathological significance and prognostic value of BZRAP1-AS1 were analyzed by using our cohort and TCGA cohort. Finally, the effect of BZRAP1-AS1 on proliferation and motility of NSCLC cell lines were evaluated by cell growth assay, colony formation assay, xenograft tumorigenesis experiment in nude mice and transwell assays respectively.ResultsCompared with adjacent normal tissues, BZRAP1-AS1 showed lower expression in NSCLC tumor tissues. As for the relationship between BZRAP1-AS1 and clinical characteristics, our results were consistent with those of TCGA data. BZRAP1-AS1 was lower in T1 than T2–T4 patients, N1-N3 than N0 patients. Low level BZRAP1-AS1 was related to shorter overall survival time (OS) in lung adenocarcinoma (LUAD), and poor first progression time (FP) in LUAD and lung squamous cell carcinoma (LUSC) patients. BZRAP1-AS1 was significantly associated with the prognosis of NSCLC patients. Overexpression of BZRAP1-AS1 inhibited proliferation and migration of H1299 and HCC827 cells.ConclusionBZRAP1-AS1 expression decreases in tumor tissues with the increase of malignancy grades in NSCLC. BZRAP1-AS1 plays an anticancer role by inhibiting cell proliferation, invasion, and metastasis, and has a potential prognostic value in NSCLC. BZRAP1-AS1 may serve as a diagnostic marker and therapeutic target for NSCLC.

RP11-79H23.3 Inhibits the Proliferation and Metastasis of Non-small-cell Lung Cancer Through Promoting miR-29c

Evidences indicate that long non-coding RNAs (lncRNAs) are closely involved and contributed to tumorigenesis and cancer progression. As a novel lncRNA, RP11-79H23.3 was found to be an anti-oncogene in bladder cancer. However, the essential roles and functions of RP11-79H23.3 in non-small-cell lung cancer (NSCLC) remains to be elucidated. Here, loss of functional assay was applied to gain insights into the functions of RP11-79H23.3 on the proliferation and metastasis capabilities of A549 and H1299 cells. Meantime, Real-time PCR was utilized to measure RP11-79H23.3 and miR-29c expression in NSCLC tissues. Dual-luciferase reporter assay, CCK8, colony formation assay, transwell and Western blot were performed to illustrate the potential molecular basis of RP11-79H23.3 in NSCLC. RP11-79H23.3 downregulation facilitated cell proliferation, migration, and invasion of NSCLC. The result of dual-luciferase reporter assay represented a direct interaction of RP11-79H23.3 with miR-29c, which suppressed miR-29c expression that showed inversely correlation in NSCLC. Moreover, RP11-79H23.3 siRNA facilitated the progression of NSCLC partially via regulating the expression of miR-29c and the activation of Wnt/β-catenin signaling pathway. Our findings highlighted that RP11-79H23.3, served as an anti-oncogene, accelerated NSCLC progression through sequestering miR-29c, providing a promising therapeutic target for NSCLC.

Tannic Acid Exhibits Antiangiogenesis Activity in Nonsmall-Cell Lung Cancer Cells.

Nonsmall-cell lung cancer (NSCLC) is the most common type of lung cancer, with a dismal prognosis. NSCLC is a highly vascularized tumor, and chemotherapy is often hampered by the development of angiogenesis. Therefore, suppression of angiogenesis is considered a potential treatment approach. Tannic acid (TA), a natural polyphenol, has been demonstrated to have anticancer properties in a variety of cancers; however, its angiogenic properties have yet to be studied. Hence, in the current study, we investigated the antiproliferative and antiangiogenic effects of TA on NSCLC cells. The (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay revealed that TA induced a dose- and time-dependent decrease in the proliferation of A549 and H1299 cells. However, TA had no significant toxicity effects on human bronchial epithelial cells. Clonogenicity assay revealed that TA suppressed colony formation ability in NSCLC cells in a dose-dependent manner. The anti-invasiveness and antimigratory potential of TA were confirmed by Matrigel and Boyden chamber studies, respectively. Importantly, TA also decreased the ability of human umbilical vein endothelial cells (HUVEC) to form tube-like networks, demonstrating its antiangiogenic properties. Extracellular vascular endothelial growth factor (VEGF) release was reduced in TA-treated cells compared to that in control cells, as measured by the enzyme-linked immunosorbent assay (ELISA). Overall, these results demonstrate that TA can induce antiproliferative and antiangiogenic effects against NSCLC.

MPZL1 upregulation promotes tumor metastasis and correlates with unfavorable prognosis in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC), accounting for 85% of all lung cancer, is one of the leading causes of cancer-related death worldwide. Previously, we demonstrated that MPZL1 gene amplification promotes liver cancer metastasis through activating Src/Cortactin pathway. However, the clinical relevance and biological roles of the MPZL1 gene in lung cancer are still unknown. Here, we found that MPZL1 expression upregulates in human NSCLC, which is partly due to the copy number amplification of this gene. Next, we observed that high MPZL1 expression correlates with unfavorable prognosis of NSCLC patients. We further demonstrated that ectopic MPZL1 overexpression promotes in vitro migratory but not proliferation and colony formation abilities of both H1299 and H460 cells. Consistently, we found that MPZL1 knockdown impairs the migratory abilities of A549 and H1775 cells. Moreover, we found that MPZL1 knockdown inhibits in vivo metastatic but not tumor growth abilities of the A549 cells. Additionally, a total of 297 differentially expressed genes (DEGs) were identified by RNA sequencing in A549 cells upon MPZL1 knockdown. By integrative analysis of DEGs regulated by MPZL1 in A549 cells and human NSCLC tissues, we revealed that COL11A1 is the potential effector gene that positively regulated by MPZL1 and correlates with poor prognosis of NSCLC patients. In conclusion, our work indicates that one of the mechanisms by which MPZL1 promotes NSCLC metastasis is through upregulating the COL11A1, and MPZL1 can be used as a biomarker to predict the prognosis of NSCLC patients.

Abstract 882: A novel p73 C-terminal isoform, p73α1, regulates inflammation and tumor suppression via the Notch1 pathway

Abstract p73, a member of the p53 family, is expressed as two N-terminal isoforms: TA, a tumor suppressor and ΔN, an oncoprotein. Alternative RNA splicing at the 3` end gives rise to multiple C-terminal isoforms, but their function is largely unknown. To address this, CRISPR was used to knock out exon 12 (E12) in several cell lines and mice, leading to isoform switching from p73α to p73α1. p73α1 is a novel isoform whose expression we detected in multiple cancer cell lines. We found that knockout of E12 inhibits the cell proliferation and migration abilities of H1299 and MIA PaCa-2 cancer cells and E12-/- MEFs exhibit increased cellular senescence. Similarly, ectopic expression of p73α1 in H1299 cells suppresses cell proliferation and knockdown of p73α1 restores the cell proliferative and migratory capacities of H1299 E12-/- cells. Consistently, we found that E12+/- mice are not prone to spontaneous tumors, unlike Trp73+/- mice, but instead are prone to chronic systemic inflammation and show elevated IL-1β and IL-6 expression in inflamed tissues. We then identified Notch1, a master regulator of the inflammatory response, as a mediator of p73α1-induced inflammation and tumor suppression, and found it to be elevated in inflamed E12+/- mouse tissues. Furthermore, we found that knockdown of Notch1 enhances cell proliferation and migration in H1299 E12-/- cells. Together, these data suggest that p73α1 plays a role in the inflammatory response and tumor suppression via Notch1. Citation Format: Kyra Laubach, Wenshen Yan, Xiangmudong Kong, Mingyi Chen, Jin Zhang, Xinbin Chen. A novel p73 C-terminal isoform, p73α1, regulates inflammation and tumor suppression via the Notch1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 882.

Activation of circ_0072088/miR-1261/PIK3CA pathway accelerates lung adenocarcinoma progression.

BackgroundCircular RNAs (circRNAs) are involved in the tumorigenesis and progression of lung adenocarcinoma (LUAD). This study aimed to determine the role of circ_0072088 in LUAD.MethodsThe existence and expression of circ_0072088 in human LUAD tissues and cell lines were determined through Sanger sequencing, quantitative reverse transcription‐polymerase chain reaction, and fluorescence in situ hybridization (FISH). Subsequently, the biological role of circ_0072088 was examined using loss‐of‐function assays in H1299 cells. Moreover, circ_0072088/miR‐1261/PIK3CA pathway‐mediated biological effects in H1299 were verified using bioinformatic prediction and experiments, including interaction analysis (FISH, luciferase reporter, and RNA‐pulldown assays), and tumor biological function test (CCK8 and colony formation, wound healing, and transwell assays). Finally, miR‐1261 and PIK3CA expression and LUAD patient survival were further analyzed using FISH, immunohistochemical staining, and the Kaplan–Meier plotter database, respectively.ResultsFirst, an increase in circ_0072088 was confirmed in human LUAD tissues. Thereafter, it was mainly localized in the cytoplasm and was found to enhance cell proliferation, migration, and invasion of H1299 cells. Mechanistically, circ_0072088 directly downregulated miR‐1261 expression, whereas increased PIK3CA gene expression was associated with poor overall survival of LUAD patients. The activation of the circ_0072088/miR‐1261/PIK3CA regulatory pathway may play a significant role in the tumorigenesis and progression of LUAD.ConclusionsCirc_0072088‐dependent regulation of miR‐1261/PIK3CA is important for cell proliferation, migration, and invasion during the tumorigenesis and progression of LUAD, warranting the need to consider the circ_0072088/miR‐1261/PIK3CA regulatory pathway as a potential therapeutic target in patients with lung adenocarcinoma.