Exploring and comparing of the gene expression and methylation differences between lung adenocarcinoma and squamous cell carcinoma

Lung cancer is one of the most frequently diagnosed malignant tumors and the main reason for cancer-related death around the world, whereas nonsmall cell lung cancer that consists two subtypes: lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) is responsible for an estimated 85% of all lung cancers. The current study aimed to explore gene expression and methylation differences between LUAD and LUSC. EdgeR was used to identify differentially regulated genes between normal and cancer in the LUAD and LUSC extracted from The Cancer Genome Atlas (TCGA), respectively, whereas SAM was used to find genes with differential methylation between normal and cancer in the LUAD and LUSC, respectively. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to analyze the function which these genes enriched in. A total of 391 genes with opposite methylation patterns in LUAD and LUSC and four functional pathways were obtained (false discovery rate (FDR) < 0.1). These pathways mainly included fat digestion and absorption, phenylalanine metabolism, bile secretion, and so on, which were related to the airframe nutrition metabolic pathway. Moreover, two genes CTSE (cathepsin E) and solute carrier family 5 member 7 (SLC5A7) were also found, among which CTSE was overexpressed and hypomethylated in LUAD corresponding to normal lung tissues, whereas SLC5A7 showed the opposite in LUAD. In conclusion, this study investigated the differences between the gene expression and methylation patterns in LUAD and LUSC, and explored their different biological characteristics. Further understanding of these differences may promote the discovery and development of new, accurate strategies for the prevention, diagnosis, and treatment of lung cancer.     

In silico assessment of EpCAM transcriptional expression and determination of the prognostic biomarker for human lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC)

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein which is involved in cell signaling, proliferation, maturation, and movement, all of which are crucial for the proper development of cells and tissues. Cleavage of the EpCAM protein leads to the up-regulation of c-myc, e-fabp, and cyclins A and E which promote tumorigenesis. EpCAM can act as potential diagnostic and prognostic biomarker for different types of cancers as it is also found to be expressed in epithelia and epithelial-derived neoplasms. Hence, we aimed to analyze the EpCAM gene expression and any associated feedback in the patients of two major types of lung cancer (LC) i.e., lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), based on the publicly available online databases. In this study, server-based gene expression analysis represents the up-regulation of EpCAM in both LUAD and LUSC subtypes as compared to the corresponding normal tissues. Besides, the histological sections revealed the over-expression of EpCAM protein in cancerous tissues by depicting strong staining signals. Furthermore, mutation analysis suggested missense as the predominant type of mutation both in LUAD and LUSC in the EpCAM gene. A significant correlation (P-value < 0.05) between the higher EpCAM expression and lower patient survival was also found in this study. Finally, the co-expressed genes were identified with their ontological features and signaling pathways associated in LC development. The overall study suggests EpCAM to be a significant biomarker for human LC prognosis.     

Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer

The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2′-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.     

The increased expression and aberrant methylation of SHC1 in non–small cell lung cancer: Integrative analysis of clinical and bioinformatics databases

Despite the previous evidence showing that SHC adaptor protein 1 (SHC1) could encode three distinct isoforms (p46SHC, p52SHC and p66SHC) that function in different activities such as regulating life span and Ras activation, the precise underlying role of SHC1 in lung cancer also remains obscure. In this study, we firstly found that SHC1 expression was up-regulated both in lung adenocarcinoma (LUAD) and in lung squamous cell carcinoma (LUSC) tissues. Furthermore, compared to patients with lower SHC1 expression, LUAD patients with higher expression of SHC1 had poorer overall survival (OS). Moreover, higher expression of SHC1 was also associated with worse OS in patients with stages 1 and 2 but not stage 3 lung cancer. Significantly, the analysis showed that SHC1 methylation level was associated with OS in lung cancer patients. It seemed that the methylation level at specific probes within SHC1 showed negative correlations with SHC1 expression both in LUAD and in LUSC tissues. The LUAD and LUSC patients with hypermethylated SHC1 at cg12473916 and cg19356022 probes had a longer OS. Therefore, it is reasonable to conclude that SHC1 has a potential clinical significance in LUAD and LUSC patients.     

The genomic alterations of lung adenocarcinoma and lung squamous cell carcinoma can explain the differences of their overall survival rates

In the US, lung carcinoma accounted for over 150,000 deaths in 2018 and the advances in increasing survival rates are still limited. In this study, we investigated the cohorts with lung adenocarcinoma (LUAD) or lung squamous cell carcinoma (LUSC) from The Cancer Genome Atlas to figure out the risk factors and genomic alterations that affected their prognosis. The histoclinical factors that differed between LUAD and LUSC were identified and the risk factors affecting the overall survival were figured out for both LUAD and LUSC. Next, the patterns of nucleotides substitutions and the mutational signatures were extracted to illustrate whether different mutational processes performed for them. Finally, the genes that had different frequencies of mutation were identified. LUAD and LUSC presented differences in histoclinical factors including age at the time of diagnosis, sex, smoking history, pathological T classification, and overall survival. This was caused by the distinct genomic alterations including the transition-to-transversion ratios, mutational signatures, and the frequently mutated genes. We proposed that the mutational signature associated with aging could be used to predict the prognosis of patients with LUAD. On the other hand, the AID/APOBEC family was associated with the prognosis of LUSC. Finally, SNTG1 and LRRK2 might be important in LUAD and LUSC, respectively.     

Comprehensive analysis of the long noncoding RNA HOXA11-AS gene interaction regulatory network in NSCLC cells

Background

Long noncoding RNAs (lncRNAs) are related to different biological processes in non-small cell lung cancer (NSCLC). However, the possible molecular mechanisms underlying the effects of the long noncoding RNA HOXA11-AS (HOXA11 antisense RNA) in NSCLC are unknown.

Methods

HOXA11-AS was knocked down in the NSCLC A549 cell line and a high throughput microarray assay was applied to detect changes in the gene profiles of the A549 cells. Bioinformatics analyses (gene ontology (GO), pathway, Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses) were performed to investigate the potential pathways and networks of the differentially expressed genes. The molecular signatures database (MSigDB) was used to display the expression profiles of these differentially expressed genes. Furthermore, the relationships between the HOXA11-AS, de-regulated genes and clinical NSCLC parameters were verified by using NSCLC patient information from The Cancer Genome Atlas (TCGA) database. In addition, the relationship between HOXA11-AS expression and clinical diagnostic value was analyzed by receiver operating characteristic (ROC) curve.

Results

Among the differentially expressed genes, 277 and 80 genes were upregulated and downregulated in NSCLC, respectively (fold change ≥2.0, P < 0.05 and false discovery rate (FDR) < 0.05). According to the degree of the fold change, six upregulated and three downregulated genes were selected for further investigation. Only four genes (RSPO3, ADAMTS8, DMBT1, and DOCK8) were reported to be related with the development or progression of NSCLC based on a PubMed search. Among all possible pathways, three pathways (the PI3K-Akt, TGF-beta and Hippo signaling pathways) were the most likely to be involved in NSCLC development and progression. Furthermore, we found that HOXA11-AS was highly expressed in both lung adenocarcinoma and squamous cell carcinoma based on TCGA database. The ROC curve showed that the area under curve (AUC) of HOXA11-AS was 0.727 (95% CI 0.663–0.790) for lung adenocarcinoma and 0.933 (95% CI 0.906–0.960) for squamous cell carcinoma patients. Additionally, the original data from TCGA verified that ADAMTS8, DMBT1 and DOCK8 were downregulated in both lung adenocarcinoma and squamous cell carcinoma, whereas RSPO3 expression was upregulated in lung adenocarcinoma and downregulated in lung squamous cell carcinoma. For the other five genes (STMN2, SPINK6, TUSC3, LOC100128054, and C8orf22), we found that STMN2, TUSC3 and C8orf22 were upregulated in squamous cell carcinoma and that STMN2 and USC3 were upregulated in lung adenocarcinoma. Furthermore, we compared the correlation between HOXA11-AS and de-regulated genes in NSCLC based on TCGA. The results showed that the HOXA11-AS expression was negatively correlated with DOCK8 in squamous cell carcinoma (r = ?0.124, P = 0.048) and lung adenocarcinoma (r = ?0.176, P = 0.005). In addition, RSPO3, ADAMTS8 and DOCK8 were related to overall survival and disease-free survival (all P < 0.05) of lung adenocarcinoma patients in TCGA.

Conclusions

Our results showed that the gene profiles were significantly changed after HOXA11-AS knock-down in NSCLC cells. We speculated that HOXA11-AS may play an important role in NSCLC development and progression by regulating the expression of various pathways and genes, especially DOCK8 and TGF-beta pathway. However, the exact mechanism should be verified by functional experiments.

     

Systems biology analysis reveals new insights into invasive lung cancer

Background

Adenocarcinoma in situ (AIS) is a pre-invasive lesion in the lung and a subtype of lung adenocarcinoma. The patients with AIS can be cured by resecting the lesion completely. In contrast, the patients with invasive lung adenocarcinoma have very poor 5-year survival rate. AIS can develop into invasive lung adenocarcinoma. The investigation and comparison of AIS and invasive lung adenocarcinoma at the genomic level can deepen our understanding of the mechanisms underlying lung cancer development.

Results

In this study, we identified 61 lung adenocarcinoma (LUAD) invasive-specific differentially expressed genes, including nine long non-coding RNAs (lncRNAs) based on RNA sequencing techniques (RNA-seq) data from normal, AIS, and invasive tissue samples. These genes displayed concordant differential expression (DE) patterns in the independent stage III LUAD tissues obtained from The Cancer Genome Atlas (TCGA) RNA-seq dataset. For individual invasive-specific genes, we constructed subnetworks using the Genetic Algorithm (GA) based on protein-protein interactions, protein-DNA interactions and lncRNA regulations. A total of 19 core subnetworks that consisted of invasive-specific genes and at least one putative lung cancer driver gene were identified by our study. Functional analysis of the core subnetworks revealed their enrichment in known pathways and biological progresses responsible for tumor growth and invasion, including the VEGF signaling pathway and the negative regulation of cell growth.

Conclusions

Our comparison analysis of invasive cases, normal and AIS uncovered critical genes that involved in the LUAD invasion progression. Furthermore, the GA-based network method revealed gene clusters that may function in the pathways contributing to tumor invasion. The interactions between differentially expressed genes and putative driver genes identified through the network analysis can offer new targets for preventing the cancer invasion and potentially increase the survival rate for cancer patients.

     

Identification immunophenotyping of lung adenocarcinomas based on the tumor microenvironment

The tumor immune microenvironment is heterogeneous, and its impact on treatment responses is not well understood. It is still a challenge to analyze the interaction between malignant cells and the tumor microenvironment to apply suitable immunotherapy in lung adenocarcinoma. We performed the nonnegative matrix factorization method to 513 messenger RNA expression profiles of lung adenocarcinomas (LUADs) from The Cancer Genome Atlas (TCGA) to obtain an immune-related expression pattern. Subsequently, we characterized the immune-related gene signatures and clinical and survival characteristics. We used 576 patients from Gene Expression Omnibus to confirm our findings. Of the patients in the training cohort, 51% had a high immune enrichment score, high expression of immune cell signaling, cytolytic activity, and interferon (IFN)-related signatures (all P < .05). We denoted these as the Immune Class. We further subdivided the Immune Class into two subclasses based on the tumor microenvironment. These were denoted the Active Immune Class and Exhausted Immune Class. The former showed significant IFN, T-cells, M1 macrophage signatures, and better prognosis (all P < .05), while the latter presented an exhausted immune response with activated stromal enrichment, M2 macrophage signatures, and immunosuppressive factors such as WNT/transforming growth factor-β (all P < .05). Furthermore, we predicted the response of our immunophenotypes to immunological checkpoint inhibitors (P < .05). Our findings provide a novel insight into the immune-related state of LUAD and can identify the patients who will be receptive to suitable immunotherapeutic treatments.     

肺腺癌诊断标志物筛选及免疫细胞浸润分析

用生物信息学方法筛选肺腺癌(Lung adenocarcinoma,LUAD)的诊断生物标志物,并分析肺腺癌中免疫细胞浸润情况。从GEO和TCGA数据库下载肺腺癌的表达数据集,利用R软件筛选肺腺癌与正常肺组织间的差异表达基因(DEGs),使用DAVID网站对DEGs进行GO及KEGG富集分析,使用STRING及Cytoscape等工具对DEGs构建蛋白相互作用网络并筛选hub基因;利用Kaplan-Meier法对DEGs进行生存分析,并对hub基因进行ROC分析筛选诊断生物标志物,利用GSEA预测有预后价值的基因参与的信号通路;并用Cibersort软件反卷积算法分析肺腺癌中免疫细胞浸润情况。共得到肺腺癌的234个DEGs,这些基因主要参与信号转导、物质代谢、免疫反应等相关信号通路;构建PPI网络筛选出的20个hub基因中8个存在预后价值(CCNA2、DLGAP5、HMMR、MMP1、MMP9、MMP13、SPP1、TOP2A),ROC分析中DLGAP5、SPP1值分别是0.703、0.706;DLGAP5、SPP1基因表达水平与肺腺癌组织浆细胞、未活化的CD4⁺记忆细胞、调节T细胞、巨噬细胞M0、M1、M2及中性粒细胞浸润密切相关(P＜0.05)。肺腺癌中DLGAP5、SPP1具有较高诊断价值且参与肺腺癌组织免疫细胞浸润;DLGAP5、SPP1基因可作为肺腺癌诊断的生物标志物,可为肺腺癌的靶向治疗研究提供新思路。     

Secreted frizzled related protein 1 is a paracrine modulator of epithelial branching morphogenesis, proliferation, and secretory gene expression in the prostate

Previous in vitro studies identified secreted frizzled related protein 1 (SFRP1) as a candidate pro-proliferative signal during prostatic development and cancer progression. This study determined the in vivo roles of SFRP1 in the prostate using expression studies in mice and by creating loss- and gain-of-function mouse genetic models. Expression studies using an Sfrp1^lacZ knock-in allele showed that Sfrp1 is expressed in the developing mesenchyme/stroma of the prostate. Nevertheless, Sfrp1 null prostates exhibited multiple prostatic developmental defects in the epithelium including reduced branching morphogenesis, delayed proliferation, and increased expression of genes encoding prostate-specific secretory proteins. Interestingly, over-expression of SFRP1 in the adult prostates of transgenic mice yielded opposite effects including prolonged epithelial proliferation and decreased expression of genes encoding secretory proteins. These data demonstrated a previously unrecognized role for Sfrp1 as a stromal-to-epithelial paracrine modulator of epithelial growth, branching morphogenesis, and epithelial gene expression. To clarify the mechanism of SFRP1 action in the prostate, the response of WNT signaling pathways to SFRP1 was examined. Forced expression of SFRP1 in prostatic epithelial cells did not alter canonical WNT/β-catenin signaling or the activation of CamKII. However, forced expression of SFRP1 led to sustained activation of JNK, and inhibition of JNK activity blocked the SFRP1-induced proliferation of prostatic epithelial cells, suggesting that SFRP1 acts through the non-canonical WNT/JNK pathway in the prostate.     

CAFET algorithm reveals Wnt/PCP signature in lung squamous cell carcinoma

We analyzed the gene expression patterns of 138 Non-Small Cell Lung Cancer (NSCLC) samples and developed a new algorithm called Coverage Analysis with Fisher's Exact Test (CAFET) to identify molecular pathways that are differentially activated in squamous cell carcinoma (SCC) and adenocarcinoma (AC) subtypes. Analysis of the lung cancer samples demonstrated hierarchical clustering according to the histological subtype and revealed a strong enrichment for the Wnt signaling pathway components in the cluster consisting predominantly of SCC samples. The specific gene expression pattern observed correlated with enhanced activation of the Wnt Planar Cell Polarity (PCP) pathway and inhibition of the canonical Wnt signaling branch. Further real time RT-PCR follow-up with additional primary tumor samples and lung cancer cell lines confirmed enrichment of Wnt/PCP pathway associated genes in the SCC subtype. Dysregulation of the canonical Wnt pathway, characterized by increased levels of β-catenin and epigenetic silencing of negative regulators, has been reported in adenocarcinoma of the lung. Our results suggest that SCC and AC utilize different branches of the Wnt pathway during oncogenesis.     

SFRP2 enhances the osteogenic differentiation of apical papilla stem cells by antagonizing the canonical WNT pathway

Background

Exploring the molecular mechanisms underlying directed differentiation is helpful in the development of clinical applications of mesenchymal stem cells (MSCs). Our previous study on dental tissue-derived MSCs demonstrated that secreted frizzled-related protein 2 (SFRP2), a Wnt inhibitor, could enhance osteogenic differentiation in stem cells from the apical papilla (SCAPs). However, how SFRP2 promotes osteogenic differentiation of dental tissue-derived MSCs remains unclear. In this study, we used SCAPs to investigate the underlying mechanisms.

Methods

SCAPs were isolated from the apical papilla of immature third molars. Western blot and real-time RT-PCR were applied to detect the expression of β-catenin and Wnt target genes. Alizarin Red staining, quantitative calcium analysis, transwell cultures and in vivo transplantation experiments were used to study the osteogenic differentiation potential of SCAPs.

Results

SFRP2 inhibited canonical Wnt signaling by enhancing phosphorylation and decreasing the expression of nuclear β-catenin in vitro and in vivo. In addition, the target genes of the Wnt signaling pathway, AXIN2 (axin-related protein 2) and MMP7 (matrix metalloproteinase-7), were downregulated by SFRP2. WNT1 inhibited the osteogenic differentiation potential of SCAPs. SFRP2 could rescue this WNT1-impaired osteogenic differentiation potential.

Conclusions

The results suggest that SFRP2 could bind to locally present Wnt ligands and alter the balance of intracellular Wnt signaling to antagonize the canonical Wnt pathway in SCAPs. This elucidates the molecular mechanism underlying the SFRP2-mediated directed differentiation of SCAPs and indicates potential target genes for improving dental tissue regeneration.

     

INHA acts as a novel and potential biomarker in lung adenocarcinoma and shapes the immune-suppressive tumor microenvironment

BackgroundINHA expression has been correlated with the development, growth, and progression of multiple cancer types. However, the biological role of INHA has not been investigated in patients with lung adenocarcinoma (LUAD). Here, we performed a comprehensive bioinformatics analysis of the LUAD dataset to determine the mechanisms underlying the regulation of tumorigenesis by INHA.Materials and methodsINHA expression and clinical information of patients with LUAD were obtained from The Cancer Genome Atlas (TCGA) database. Protein levels in LUAD cell lines and human lung epithelial cells were examined by western blotting. Next, the prognostic value of INHA in LUAD was assessed using Cox regression analysis, while the potential biological functions and the impact on the immune microenvironment of INHA were investigated using gene set enrichment analysis (GSEA) and single sample GSEA (ssGSEA). Finally, the effect of INHA on LUAD cell proliferation and invasion was determined in vitro and in vivo.ResultsWe found significantly high mRNA and protein expression levels of INHA in LUAD tissues and cell lines. Additionally, a higher expression of INHA was linked to a shorter overall survival (OS) and a worse pathological stage, while INHA expression was associated with immune cell infiltration and immune-related markers in the LUAD tumor microenvironment. LUAD with high INHA expression tends to be a cold tumor. Furthermore, GO and KEGG enrichment analysis indicated that INHA-related genes were enriched in the cell adhesion and immune signaling pathways of LUAD. INHA promoted LUAD cell proliferation and invasion, in vitro and in vivo, by inducing the EGFR pathway.ConclusionOur findings revealed that INHA is overexpressed in LUAD and is linked to a poor prognosis. Our study demonstrates the potential of INHA as an immunotherapeutic and predictive biomarker in LUAD.