Promotes Lung Cancer Cell Growth Research Articles

Activation of Hypoxia Inducible Factor-1 Alpha-Mediated DNA Methylation Enzymes (DNMT3a and TET2) Under Hypoxic Conditions Regulates S100A6 Transcription to Promote Lung Cancer Cell Growth and Metastasis.

Aims: This research was aimed at investigating the effects of hypoxia inducible factor-1 alpha (HIF-1α)-mediated DNA methylation enzymes (ten-eleven translocase-2 [TET2] and DNA methyltransferase-3a [DNMT3a]) under hypoxic conditions on S100A6 transcription, thereby promoting the growth and metastasis of lung cancer cells. Methods: The expression of HIF-1α or S100A6 in lung cancer cells was interfered with under normoxic and hypoxic conditions, and the cell proliferative, migratory, and invasive properties were assessed. The mechanism of HIF-1α-regulated TET2 and DNMT3 effects on S100A6 transcription under hypoxic conditions was further investigated. Results: Functionally, S100A6 over-expression promoted lung cancer cell proliferation and metastasis. S100A6 over-expression reversed the inhibitory effects of HIF-1α interference on the proliferation and metastasis of lung cancer cells. S100A6 was induced to express in an HIF-1α-dependent manner under hypoxic conditions, and silencing S100A6 or HIF-1α suppressed lung cancer cell proliferation and metastasis under hypoxic conditions. Further, The Cancer Genome Atlas-lung adenocarcinoma database analysis revealed that S100A6 mRNA levels had a negative correlation with methylation levels. Mechanistically, CpG hypomethylation status in the S100A6 promoter hypoxia response element had an association with HIF-1α induction. TET2 was enriched in S100A6 promoter region of lung cancer cells under hypoxic conditions, whereas DNMT3a enrichment was reduced in S100A6 promoter region. HIF-1α-mediated S100A6 activation was linked to DNMT3a-associated epigenetic inactivation and TET2 activation. Innovation: The activation of HIF-1α-mediated DNA methylation enzymes under hypoxic conditions regulated S100A6 transcription, thereby promoting lung cancer cell growth and metastasis. Conclusion: In lung cancer progression, hypoxia-induced factor HIF-1α combined with DNA methylation modifications co-regulates S100A6 transcriptional activation and promotes lung cancer cell growth and metastasis.

MiR-196b-5p activates NF-κB signaling in non-small cell lung cancer by directly targeting NFKBIA

BackgroundOur recent study found that QKI-5 regulated miRNA, miR-196b-5p, promotes non-small cell lung cancer (NSCLC) progression by directly targeting GATA6, TSPAN12 and FAS. However, the biological functions of miR-196b-5p in NSCLC progression and metastasis still remain elusive. MethodsCell proliferation, migration, colony formation, cell cycle assays were used to investigate cellular phenotypic changes. Quantitative real-time PCR (qRT-PCR) and western blot analyses were used to measure expressions of relative gene and protein. Interaction between QKI-5 and miR-196b-5p was determined by RNA immunoprecipitation (RIP) assay. Luciferase reporter assay was used to determine direct binding between miR-196b-5p and NFKBIA 3′-UTR. ELISA assay was used to measure secreted IL6 proteins. Mice xenograft model was used to assess the functions of NFKBIA on in vivo tumor growth. ResultsWe demonstrated that the miR-196b-5p facilitates lung cancer cell proliferation, migration, colony formation, and cell cycle by directly targeting NFKBIA, a negative regulator of NFκB signaling. Knocking down NFKBIA increases IL6 mediated phosphorylation of signal transducer and activator of transcription 3 (STAT3) to promote lung cancer cell growth by activating NFκB signaling. The expression of NFKBIA was significantly downregulated in NSCLC tissue samples, and was negatively correlated with the expression miR-196b-5p. In addition, we found that downregulated QKI-5 expression was associated with the elevated miR-224 expression in NSCLC. ConclusionsOur findings indicated that the miR-224/QKI-5/miR-196b-5p/NFKBIA signaling pathway might play important functions in the progression of NSCLC, and suggested that targeting this pathway might be an effective therapeutic strategy in treating NSCLC.

Characterization of URST1 as a prognostic marker and therapeutic target for lung cancer.

e21000 Background: The improved understanding of cancer-specific oncoproteins could lead to the development of novel biomarkers or therapeutic targets for lung cancers. Methods: We screened oncoproteins as follows: i) Identification of up-regulated genes in lung cancers by cDNA microarray, ii) Validation of clinicopathologic importance of their gene product by tissue microarray, iii) Examination of the growth of cancer cells after transfection of their siRNA or expression vector, iv) Screening of their signal pathways. Results: We identified up-regulated in solid tumor 1 (URST1) as a candidate. Immunohistochemical staining of URST1 showed that URST1 expression was observed in 231 (64.5%) of 358 non-small cell lung cancers (NSCLC) and 11 (84.6%) of 13 small cell lung cancers. High level of URST1 expression was associated with poor prognosis for NSCLC patients ( P = 0.0003). Multivariate analysis confirmed that strong URST1 expression was an independent prognostic factor for NSCLC patients that had undergone radical surgery. Knockdown of URST1expression by siRNAs against URST1 significantly suppressed the growth of lung cancer cells, and also inhibited the cell invasive ability. Treatment of lung cancer cells with small molecule inhibitor against URST1 suppressed the cell growth partly through G2/M arrest and cell death. In contrast, enforced expression of URST1 significantly promoted lung cancer cell growth. Microarray analysis coupled with siRNAs for URST1 suggested various URST1 related oncogenic pathways in lung cancer cells. Conclusions: URST1 could play an essential role in malignant potential of lung cancer and it might be useful as a prognostic biomarker and therapeutic target molecule for lung cancer.

Exosomes from cisplatin-induced dormant cancer cells facilitate the formation of premetastatic niche in bone marrow through activating glycolysis of BMSCs.

Lung cancer is the leading cause of cancer-related deaths worldwide. Chemotherapy kills most cancer cells; however, residual cells enter a dormant state. The dormant cancer cells can be reactivated under specific circumstances. The "premetastatic niche" that is suitable for colonization of cancer cells is formed before the arrival of cancer cells. Tumor-derived exosomes are the main mediators of tumorigenesis. We are aiming to elucidate the roles of exosomes from cisplatin-induced dormant lung cancer cells in the formation of premetastatic niches in bone marrow. We performed differential proteomics in dormant A549 cell- and A549 cell-derived exosomes. Non-targeted metabolomics and RNA sequencing were performed to explore the molecular and metabolic reprogramming of bone marrow stromal cells (BMSCs). The growth and metastasis of A549 cells in vivo were monitored by bioluminescence imaging. We found that Insulin-like growth factor 2 (IGF-2) and Insulin-like growth factor binding protein 2 (IGFBP2) were upregulated in dormant A549 cell-derived exosomes. BMSCs that took up exosomes from dormant A549 cells showed enhanced glycolysis and promoted the growth and metastasis of A549 cells possibly through Insulin-like growth factor 1 receptor (IGF-1R)-induced metabolic reprogramming. Inhibition of the production of lactate and IGF-1R signaling can suppress the growth and metastasis of A549 cells from bone marrow. Overall, we demonstrated that BMSCs formed a premetastatic niche upon taking up exosomes from cisplatin-induced dormant lung cancer cells. BMSCs promoted lung cancer cell growth and metastasis through the reverse Warburg effect.

Kruppel like factor 16 promotes lung adenocarcinoma progression by upregulating lamin B2

ABSTRACT Lung cancer is one of the most common causes of cancer-related death. In the past decade, the treatment and diagnosis of lung cancer have progressed significantly in early efforts to promote the survival of lung cancer patients. Kruppel like factor 16 (KLF16) is a zinc finger transcription factor that regulates a diverse array of developmental events and cellular processes. KLF16 is involved in the progression of various cancer types. However, the role of KLF16 in the development of lung cancer remains unknown. In this study, KLF16 was overexpressed in lung cancer samples. KLF16 downregulation inhibited lung cancer cell proliferation and migration. Conversely, KLF16 overexpression promoted lung cancer cell growth and invasion. Mechanistically, the expression level LMNB2 was suppressed by KLF16 knockdown and was promoted by KLF16 overexpression. The overall survival of patients with high LMNB2 levels was poor. Luciferase assays showed that KLF16 promoted the transcription activity of LMNB2 gene. Concomitantly, the expression level of LMNB2 was also higher in lung adenocarcinoma (LUAD) than in normal tissues, and its knockdown or overexpression can reverse the effect of KLF16 overexpression or knockdown on lung cancer cell proliferation, migration, and even tumorigenesis, indicating that LMNB2 also functions as an oncogene. In conclusion, KLF16 can be used as a potential therapeutic and preventive biomarker in lung cancer treatment and prognosis by actively regulating the expression of LMNB2.

DNAJC12 activated by HNF1A enhances aerobic glycolysis and drug resistance in non-small cell lung cancer.

BackgroundThe DnaJ heat shock protein family member C12 (DNAJC12) gene has been demonstrated to promote lung cancer cell growth and migration by increasing β‑catenin expression. In this study, we aimed to reveal the role of DNAJC12 in modulating aerobic glycolysis and cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC).MethodsBioinformatics analysis was applied to assess the expression levels of DNAJC12 and hepatocyte nuclear factor 1-alpha (HNF1A) in NSCLC tissues and the correlation between DNAJC12 and HNF1A expression levels. Cell Counting Kit-8 (CCK-8), flow cytometry assay, and in vivo tumor formation were used to evaluate cell growth, apoptosis, and tumorigenesis. Luciferase gene reporter assay was adopted to assess the relationship between HNF1A and DNAJC12.ResultsBoth DNAJC12 and HNF1A were overexpressed in NSCLC tissues and cells, and their expressions showed a positive correlation. The HNF1A gene could bind to the promoter of DNAJC12 and promoted its transcription and translation. Overexpression of both DNAJC12 and HNF1A promoted cell growth, aerobic glycolysis, and inhibited cell apoptosis in NCI-H1975 and NCI-H1650 cells, as well as the cell apoptosis induced by DDP. In addition, cell growth and aerobic glycolysis mediated DNAJC12 were reversed by the silencing of β-catenin, and downregulation of DNAJC12 abolished the above roles of HNF1A.ConclusionsThis study revealed that DNAJC12, activated by the transcription factor HNF1A, could enhance aerobic glycolysis and drug resistance to DDP in NSCLC through regulating β-catenin expression.

KRT6A Promotes Lung Cancer Cell Growth and Invasion Through MYC-Regulated Pentose Phosphate Pathway.

Keratin 6A (KRT6A) belongs to the keratin protein family which is a critical component of cytoskeleton in mammalian cells. Although KRT6A upregulation in non-small cell lung cancer (NSCLC) has been reported, the regulatory mechanism and functional role of KRT6A in NSCLC development have been less well investigated. In this study, KRT6A was confirmed to be highly expressed in NSCLC tissue samples, and its high expression correlated with poor patient prognosis. Furthermore, overexpression of KRT6A promotes NSCLC cell proliferation and invasion. Mechanistically, KRT6A overexpression is sufficient to upregulate glucose-6-phosphate dehydrogenase (G6PD) levels and increase the pentose phosphate pathway flux, an essential metabolic pathway to support cancer cell growth and invasion. In addition, we discovered that lysine-specific demethylase 1A (LSD1) functions upstream to promote KRT6A gene expression. We also found that the MYC family members c-MYC/MYCN are involved in KRT6A-induced G6PD upregulation. Therefore, this study reveals an underappreciated mechanism that KRT6A acts downstream of LSD1 and functions as a pivotal driver for NSCLC progression by upregulating G6PD through the MYC signaling pathway. Together, KRT6A and LSD1 may serve as potential prognostic indictors and therapeutic targets for NSCLC.

MicroRNA-155-5p promotes cell proliferation and invasion in lung squamous cell carcinoma through negative regulation of fibroblast growth factor 9 expression.

BackgroundNon-small cell lung cancer (NSCLC) ranks first for mortality among all malignancies. Squamous cell carcinoma (SCC) is one of the main types of NSCLC. Previous studies have found that fibroblast growth factor 9 (FGF9) is closely related to lung SCC via different molecular regulatory mechanisms. This study aimed to explore the relationship between microRNA-155-5p (miR-155-5p) and FGF9 gene expression and their effects on the proliferation and invasion of lung SCC through experiments, in order to provide theoretical basis for overcoming this disease.MethodsFluorescence quantitative polymerase chain reaction was employed for the detection miR-155-5p and FGF9 expression in lung SCC tissues (n=40) and the corresponding adjacent normal tissues. The expression of FGF9 in the cancerous and adjacent tissues was detected by western blot. Transwell assay used to verify the effect of miR-155-5p on FGF-induced invasion and migration. Finally, subcutaneous tumor formation experiments in nude mice were used to verify how miR-155-5p and FGF9 affect the proliferative ability of lung SCC cells.ResultsThe results of fluorescence quantitative PCR revealed that miR-155-5p and FGF9 were expressed at high and low levels, respectively, in lung SCC tissue samples relative to normal adjacent tissue samples. Western blot analysis of 6 lung SCC tissue samples revealed a significantly reduced level of FGF9. Correlation analysis uncovered that miR-155-5p and FGF9 share a significant negative correlation in lung SCC. At the messenger RNA and protein levels miR-155-5p could negatively regulate the expression of FGF9. Bioinformatics and dual luciferase reporter assay results confirmed FGF9 to be a downstream regulatory gene targeted by miR-155-5p. Our in vitro and in vivo results demonstrated that FGF9 overexpression exerted a significant inhibitory effect on miR-155-5p’s ability to promote lung cancer cell growth, invasion, and proliferation.ConclusionsOur results show that miR-155-5p, as an oncogene, negative regulates FGF9 expression to promote SCC occurrence and development in the lungs.

M2 macrophages\u2010derived exosomal microRNA-501-3p promotes the progression of lung cancer via targeting WD repeat domain 82

BackgroundExosomes are known to transmit microRNAs (miRNAs) to affect cancer progression, while the role of M2 macrophages-derived exosomes (M2 exosomes) conveying miR-501-3p in lung cancer (LC) remains unknown. We aim to explore the role of exosomal miR-501-3p in LC development via targeting WD repeat domain 82 (WDR82).MethodsLung cancer tissue and normal tissue specimens were collected, in which tumor-associated macrophages (TAM) were measured by immunohistochemistry. M2 macrophages were induced and treated with altered miR-501-3p, and then the exosomes were extracted and identified. MiR-501-3p and WDR82 expression in LC tissues and cell liens was determined. The predictive role of miR-501-3p in prognosis of LC patients was assessed, and the proliferation, colony formation ability, invasion, migration and apoptosis of the LC cells were determined. Targeting relationship between miR-501-3p and WDR82 was confirmed.ResultsTAM level was elevated in lung cancer tissues. MiR-501-3p was upregulated while WDR82 was downregulated in LC tissues and cell lines, and the M2 exosomes further upregulated miR-501-3p. M2 exosomes and exosomal miR-501-3p promoted LC cell growth. MiR-501-3p inhibition reversed the effect of M2 exosomes on LC cells. WDR82 was confirmed as a target gene of miR-501-3p.ConclusionM2 macrophages-derived exosomal miR-501-3p promotes the progression of LC via downregulating WDR82.

LncRNA DPP10-AS1 promotes malignant processes through epigenetically activating its cognate gene DPP10 and predicts poor prognosis in lung cancer patients.

Objective:The purpose of this study was to explore the function and gene expression regulation of the newly identified lncRNA DPP10-AS1 in lung cancer, and its potential value as a prognostic biomarker.Methods:qRT-PCR and Western blot were conducted to detect the expression of DDP10-AS1 and DPP10 in lung cancer cell lines and tissues. The effects of DDP10-AS1 on DPP10 expression, cell growth, invasion, apoptosis, and in vivo tumor growth were investigated in lung cancer cells by Western blot, rescue experiments, colony formation, flow cytometry, and xenograft animal experiments.Results:The novel antisense lncRNA DPP10-AS1 was found to be highly expressed in cancer tissues (P < 0.0001), and its upregulation predicted poor prognosis in patients with lung cancer (P = 0.0025). Notably, DPP10-AS1 promoted lung cancer cell growth, colony formation, and cell cycle progression, and repressed apoptosis in lung cancer cells by upregulating DPP10 expression. Additionally, DPP10-AS1 facilitated lung tumor growth via upregulation of DPP10 protein in a xenograft mouse model. Importantly, DPP10-AS1 positively regulated DPP10 gene expression, and both were coordinately upregulated in lung cancer tissues. Mechanically, DPP10-AS1 was found to associate with DPP10 mRNA but did not enhance DPP10 mRNA stability. Hypomethylation of DPP10-AS1 and DPP10 contributed to their coordinate upregulation in lung cancer.Conclusions:These findings indicated that the upregulation of the antisense lncRNA DPP10-AS1 promotes lung cancer malignant processes and facilitates tumorigenesis by epigenetically regulating its cognate sense gene DPP10. DPP10-AS1 may serve as a candidate prognostic biomarker and a potential therapeutic target in lung cancer.

TRIP13 promotes lung cancer cell growth and metastasis through AKT/mTORC1/c-Myc signaling.

Lung adenocarcinoma (LUAD) is a primary cause of cancer-patient mortality throughout the world. Thyroid hormone receptor interactor 13 (TRIP13) is a gene that expresses a protein involved in cell division, including tumorigenesis. Its expression is high in various human tumors; however, its role in LUAD cells remains undetermined. To investigate the TRIP13's role in the development of LUAD. Bioinformation analysis was used to analyze the expression of TRIP13 in LUAD tissues and the impact on the prognosis of LUAD; CRISPR/Cas9 was used to construct the cell lines; CCK-8 was used to explore the cell proliferation; Transwell assays was applied to exam the cell migration and cell invasion abilities; Western blot and immunoprecipitation was used to explore the relation between TRIP13 and AKT/mTORC1/c-Myc signaling pathway. By analyzing LUAD data from The Cancer Genome Atlas and the Gene Expression Omnibus databases, we determined that TRIP13 is highly expressed in LUAD tissues and that this expression level has a negative impact on the patient mortality. TRIP13 has also proved to promote LUAD cell proliferation, migration, and invasion. In this study, we demonstrated that TRIP13 activates AKT/mTORC1/c-Myc signaling in these cells. Our results have identified the role and potential mechanism by which TRIP13 affects LUAD cells, which may provide a useful marker for helping to diagnose this disease and create new therapies against it.

MiR-196b-5p-mediated downregulation of FAS promotes NSCLC progression by activating IL6-STAT3 signaling

Our recent study demonstrated that the QKI-5 regulated miRNA, miR-196b-5p, and it functions as an onco-microRNA in non-small cell lung cancer (NSCLC) by directly targeting GATA6 and TSPAN12. However, the role of miR-196b-5p in NSCLC progression and metastasis still remains unclear. We found that miR-196b-5p promotes lung cancer cell proliferation and colony formation by directly targeting tumor suppressor, FAS. The expression of FAS was significantly downregulated in NSCLC tissue samples and was negatively correlated with the miR-196b-5p expression. Knocking down FAS activates NFkB signaling and subsequent IL6 secretion, resulting in phosphorylation of signal transducer and activator of transcription 3 (STAT3) to promote lung cancer cell growth. Our findings indicated that miR-196b-5p might exhibit novel oncogenic function by FAS-mediated STAT3 activation in NSCLC, and suggested that targeting the miR-196b-5p/FAS/NFkB/IL6/STAT3 pathway might be a promising therapeutic strategy in treating NSCLC.

Prospects for miR-21 as a Target in the Treatment of Lung Diseases.

MicroRNA (miRNA/miR) is a class of small evolutionarily conserved non-coding RNA, which can inhibit the target gene expression at the post-transcriptional level and serve as significant roles in cell differentiation, proliferation, migration and apoptosis. Of note, the aberrant miR-21 has been involved in the generation and development of multiple lung diseases, and identified as a candidate of biomarker, therapeutic target, or indicator of prognosis. MiR-21 relieves acute lung injury via depressing the PTEN/Foxo1-TLR4/NF-κB signaling cascade, whereas promotes lung cancer cell growth, metastasis, and chemo/radio-resistance by decreasing the expression of PTEN and PDCD4 and promoting the PI3K/AKT transduction. The purpose of this review is to elucidate the potential mechanisms of miR-21 associated lung diseases, with an emphasis on its dual regulating effects, which will trigger novel paradigms in molecular therapy.

Abstract 2525: Identification of mRNA targets associated with PIWIL1 protein complex in lung cancer cells

Abstract Introduction: In previous published work we have shown that RASSF1C promotes expression of PIWIL1 and PIWI interacting RNAs (piRNAs) in lung cancer cells, suggesting the hypothesis that that the RASSF1C-PIWI-piRNA pathway may promote lung cancer cell growth and progression. PIWIL1 protein-piRNA complexes have recently been shown to regulate mRNAs in mouse testes, suggesting that PIWIL1 plays an important role in mRNA cleavage to regulate specific targets involved in cellular growth. To investigate this hypothesis, we used a non-small cell lung cancer (NSCLC) cell model to identify candidate mRNAs targeted by the RASSF1C-PIWIL1-piRNA pathway through PIWIL1-protein complex co-immunoprecipitation and RNA sequencing. Methods: Cell lysates were prepared under non-denaturing conditions from the lung cancer cell line H1299 over-expressing RASSF1C (experimental sample) and H1299 over-expressing the vector backbone (control sample) using a co-immunoprecipitation kit (Pierce). The PIWIL1 2C12 monoclonal antibody (Wako Chemicals Inc., Richmond, VA) was used to pull down PIWIL1 protein using 1 mg of cell lysates from experimental and control samples. The RNA associated with the precipitated PIWIL1 protein complex was isolated using a total RNA isolation kit (Qiagen) and the isolated RNA was subjected to RNA deep sequencing and bioinformatic analysis. Results: Differential expression analysis based on read counts was carried out using a fold-change threshold of 2.0, maximum p-value of 0.05, and a minimum 1CPM, identified over 5505 genes that are differentially expressed when comparing read counts from H1299 cells over-expressing RASSF1C to H1299 cells over-expressing the vector back bone. Some of the genes identified with reduced mRNA levels correspond to tumor suppressors such as PTEN and p53 and apoptosis-inducing genes such as BAD and CASP3. Conclusions: Our study was designed to show that PIWIL-protein complexes can be pulled down from lung cancer cell lysates, and that PIWIL-protein complexes are likely to bind and regulate mRNA targets in lung cancer cells. We have identified interesting mRNAs that are potentially silenced by the PIWIL1-piRNA protein complex. Ongoing work is focused on validating some of the potential mRNA targets identified. This paves the way for conducting RNA-sequencing studies to identify specific piRNAs that associate with PIWIL1-protein complexes in the nucleus and cytoplasm. Citation Format: Yousef G. Amaar, Mark E. Reeves. Identification of mRNA targets associated with PIWIL1 protein complex in lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2525.

IL-17A and IL-17F orchestrate macrophages to promote lung cancer.

Previously, inflammation has been found to be associated with the development of lung cancer. Despite their well-characterized pro-inflammatory functions, the putative roles of interleukin-17 (IL-17) cytokine family members in tumorigenesis have remained controversial. While IL-17A exhibits both pro- and anti-tumor effects, IL-17F has been suggested to serve as a candidate for cancer therapy. Thus, we aimed at clarifying the involvement of IL-17A/F in lung cancer. IL-17 receptor expression in human and murine lung cancer cells was assessed using immunofluorescence. The effect of IL-17A/F stimulation on lung cancer cell viability (SRB assay) and metabolism (glucose consumption and lactate production) was evaluated under normoxic and hypoxic conditions. Characterization of IL-17A/F-stimulated macrophages was performed by flow cytometry and ELISA. The effect of conditioned media (CM) from IL-17A/F-stimulated macrophages was evaluated on lung cancer cell migration. The effect of CM-stimulated macrophages on lung tumor growth, proliferation and angiogenesis was evaluated in vivo using a chicken chorioallantoic membrane (CAM) assay. No alterations in lung cancer cell viability or metabolism were observed upon direct stimulation with IL-17A/F. We found, however, that CM from IL-17A/F-stimulated macrophages promoted both murine and human lung cancer cell progression through an increased migration capacity in vitro and enhanced in vivo tumor growth, proliferation and angiogenesis. These findings were supported by an increased polarization of human macrophages towards a M2-like phenotype. Our data indicate that IL-17A/F act through immune cell orchestration, i.e., of macrophages, to promote lung cancer cell growth and progression. In addition, our data provide a link between IL-17A/F activity and lung cancer cell-macrophage crosstalk.

PP4C facilitates lung cancer proliferation and inhibits apoptosis via activating MAPK/ERK pathway

PurposeProtein phosphatase 4 catalytic subunit (PP4C) has been shown to play crucial regulatory roles in biological process and is frequently upregulated in cancer such as breast and colorectal carcinoma. However, the function and potential molecular mechanism of PP4C in lung cancer remains unclear. MethodsBioinformatic analysis was used to detect the expression level and prognosis of patients. Western blot, quantitative real-time PCR (qRT-PCR), CCK8, 5-Ethynyl-2′-deoxyuridine (Edu) proliferation assay and flow cytometric were used to explore the function in lung cancer cells. ResultsIn this study, we found that PP4C was upregulated in lung cancer tissues as compared with that in normal lung tissues. Furthermore, patients with high expression level of PP4C were correlated with a poor prognosis in lung cancer patients. In vitro, CCK8, Edu proliferation assays and flow cytometry analysis showed that PP4C could promote lung cancer cell growth and inhibit apoptosis. Mechanistic investigations revealed that PP4C may interact with PP4R1 and promote ERK activation. Additionally, PP4C depletion resulted in lower tumor growth in vivo. ConclusionsTaken together, these data showed the oncogenic of PP4C in NSCLC tumorigenesis and provide a new insight of PP4C in the progression of NSCLC.

SNP rs17079281 Decreases Lung Cancer Risk Through Creating an YY1-Binding Site to Suppress DCBLD1 Expression

Background: Genome-wide association studies (GWAS) have identified numerous genetic polymorphisms that are associated with cancer risk, but their biological function remains largely unknown. Methods: We performed a linkage disequilibrium (LD) analysis on a GWAS-discovered SNP rs9387478 and identified several potentially functional SNPs. We examined the associations of these SNPs with lung cancer in case and control study. Luciferase assay, CHIP-qPCR and CRISPR/Cas9-mediated gene editing experiments were performed to confirm the effect of rs17079281 on gene DCBLD1 expression. Knockdown or upregulation of DCBLD1 in cancer cells were evaluated for oncogenic properties using cell-based functional assays and a mouse xenograft model. Findings: The results suggested that lung cancer risk was associated with SNP rs17079281 and that patients with genotype C/T or T/T had lower risk compared with those with genotype C/C (OR=0.78, 95% CI=0.63-0.98), which was validated with in a case-control study (558 cases and 534 controls) and a meta-analysis of 4403 cases and 5336 controls which included 3 GWASs. YY1 had higher binding-affinity to the T alleles of rs17079281 than to the C alleles, which influenced the expression of DCBLD1 in allele-specific manner. Our study further showed that DCBLD1 promoted lung cancer cell growth by influencing cell cycle. Interpretation: These findings revealed how a risk SNP rs17079281 conferred susceptibility to tumorigenesis and the important role of DCBLD1 in participating the development of lung cancer. Funding: This work was supported by the National Natural Science Foundation of China. Declaration of Interest: The authors declare no potential conflicts of interest. Ethical Approval Statement: The two case-control studies were approved by the ethic review committees at Tianjin Medical University Cancer Hospital and the institutional review board of China Medical University, respectively. Each study subject signed an informed consent before being enrolled in the study in accordance with the Declaration of Helsinki. The animal experiment was approved by the Shanghai Jiao Tong University School of Medicine Animal Ethics Committee and carried out in accordance to the approved protocols.

YTH domain family 2 promotes lung cancer cell growth by facilitating 6-phosphogluconate dehydrogenase mRNA translation

N6-methyladenosine (m6A) is one of widespread post-transcriptional mRNA modifications in eukaryotes and the m6A modification plays critical roles in various human cancers. However, the role of m6A-binding proteins in cancer metabolism remains elusive. Here, we report that YTH domain family 2 (YTHDF2) is upregulated in lung cancer tissues, promotes lung cancer cell growth and enhances the pentose phosphate pathway (PPP) flux, which is crucial for tumor growth. Mechanistically, YTHDF2 directly binds to the m6A modification site of 6-phosphogluconate dehydrogenase (6PGD) three prime untranslated region (3'-UTR) to promote 6PGD mRNA translation in lung cancer cells. Collectively, our data indicate that YTHDF2 acts as a tumor promoter to enhance tumor growth via facilitating 6PGD mRNA translation.

NCBP1 promotes the development of lung adenocarcinoma through up-regulation of CUL4B.

Lung cancer is the most frequent cancer type and is the leading cause of tumour‐associated deaths worldwide. Nuclear cap‐binding protein 1 (NCBP1) is necessary for capped RNA processing and intracellular localization. It has been reported that silencing of NCBP1 resulted in cell growth reduction in HeLa cells. Nevertheless, its clinical significance and underlying molecular mechanisms in non–small‐cell lung cancer remain unclear. In this study, we found that NCBP1 was significantly overexpressed in lung cancer tissues and several lung cancer cell lines. Through knockdown and overexpression experiments, we showed that NCBP1 promoted lung cancer cell growth, wound healing ability, migration and epithelial‐mesenchymal transition. Mechanistically, we found that cullin 4B (CUL4B) was a downstream target gene of NCBP1 in NSCLC. NCBP1 up‐regulated CUL4B expression via interaction with nuclear cap‐binding protein 3 (NCBP3). CUL4B silencing significantly reversed NCBP1‐induced tumorigenesis in vitro. Based on these findings, we propose a model involving the NCBP1‐NCBP3‐CUL4B oncoprotein axis, providing novel insight into how CUL4B is activated and contributes to LUAD progression.

AKT-mediated phosphorylation enhances protein stability and transcription activity of ZNF322A to promote lung cancer progression

ZNF322A is an oncogenic zinc-finger transcription factor. Our published results show that ZNF322A positively regulates transcription of alpha-adducin (ADD1) and cyclin D1 (CCND1) to promote tumorgenicity of lung cancer. However, the upstream regulatory mechanisms of ZNF322A protein function remain elusive. Here, we demonstrate that AKT could phosphorylate ZNF322A by in vitro kinase assay and cell-based mass spectrometry analysis. Overexpression of AKT promoted ZNF322A protein stability and transcriptional activity, whereas these effects were inhibited by knockdown of AKT or treating with AKT inhibitor. We studied AKT-mediated phosphorylation sites, viz. Thr-150, Ser-224, Thr-234, and Thr-262. ZNF322A phosphorylation at Thr-262 by AKT promoted ZNF322A protein stability thus increased ADD1 promoter activity. Interestingly, phosphorylation at Thr-150, Ser-224, and Thr-234 enhanced transcription activity without affecting protein stability of ZNF322A. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that ZNF322A phosphorylation defective mutants Thr-150A, Ser-224A, and Thr-234A attenuated chromatin binding and DNA binding affinity to ADD1 and CCND1 promoters compared with wild-type ZNF322A. Furthermore, AKT-mediated Thr-150, Ser-224, Thr-234, and Thr-262 phosphorylation promoted lung cancer cell growth and metastasis in vitro and in vivo. Clinically, expression of phosphorylated ZNF322A (p-ZNF) correlated with actively phosphorylated AKT (p-AKT) in tumor specimens from 150 lung cancer patients. Multivariate Cox regression analysis indicated that combined p-AKT and p-ZNF expression profile was an independent factor to predict the clinical outcome in lung cancer patients. Our results reveal a new mechanism of AKT signaling in promoting ZNF322A protein stability and transcriptional activity in lung cancer cell, xenograft, and clinical models.