As one of the most lethal and prevalent malignant tumors, LUAD has a high incidence and mortality rate(Relli et al., 2019). Treating LUAD has been a hot topic of clinical research in oncology for several years. Nowadays, studies have confirmed that some biomarkers are associated with metastasis, prognosis, and survival of LUAD(Zhang et al., 2019). It has laid a foundation for the diversity of lung cancer treatment in recent years. However, in order to improve the OS rate of LUAD, identifying of early biomarkers is still a major breakthrough point for targeted therapy(Lamort et al., 2022). NETs are the absolutely necessary mechanism in innate immunity, and which are believed as forefront in the immune microenvironment of tumors(Demkow, 2021; Li et al., 2023). While the biological mechanisms and prognostic biomarkers of LUAD concerning NETs are still limited(Zhang et al., 2022). There is a new study has shown that NETs-associated lncRNA signaling can predict clinical outcomes in patients with LUAD(Ding et al., 2022; Wu et al., 2022b). Inspired by the role of NETs in LUAD, we conducted this study.
In the current study, six risk model genes associated with NETs were identified, namely IGF2BP1, ABCC2, BCL2L10, FIGF, EMR3 and WNT7A. IGF2BP1 has the ability to recognize the GG(m6A) C sequence within targeted mRNAs, and thus is involved in the transcription, stability, and translation processes of various RNA molecules(Jiang et al., 2022a). Hansheng Wu et al. found that IGF2BP1 could act as a key factor in promoting cell proliferation, invasion and migration and inhibiting apoptosis in LUAD, its high expression was significantly correlated with high clinical stage and poor prognosis of LUAD patients(Wu et al., 2022a). This study further supports our study on the relationship between IGF2PB1 overexpression and prognosis in LUAD patients. As an ATP-binding cassette transporter, ABCC2 is involved in the export of endophytes and xenobiotics, which is likely to be a prognostic biomarker for LUAD through ABC family-mediated transporter dysfunction(Zhang et al., 2021). This finding is consistent with our investigation of risk model genes. BCL2L10 has oncogenic and tumor suppressive functions according to the type of cancer. It functions as a tumor suppressor gene in gastric cancer (GC) and hepatocellular carcinoma. In GC, a hypermethylated state of BCL2L10 reduced BCL2L10 expression. And meanwhile, knocking out the BCL2L10 promoted cell proliferation via activation of the PI3K-Akt signaling(Mikata et al., 2010; Xu et al., 2011). Likewise, BCL2L10 expression is downregulated in hepatocellular carcinoma, and overexpression of BCL2L10 inhibits activation of JAK-STAT signaling(Bai et al., 2017). In our study, BCL2L10 expression was increased in LUAD patients, which may be different from the above mechanism, but as an oncogene of LUAD, its specific mechanism needs further study. FIGF is a human homolog of mouse figf (c-fos-induced growth factor), and it is also a new member of platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family(Rocchigiani et al., 1998). Studies have confirmed that enhanced expression of FIGF can promote insulin expression levels and thus reduce blood glucose levels(He et al., 2019). FIGF is upregulated by Sulf2, thereby promoting breast cancer progression and regulating the expression of tumor-associated genes in breast cancer (BC)(Zhu et al., 2016). However, there is no report on the role of FIGF in LUAD, which requires further research on its mechanism. EMR3 is expressed at the highest level in neutrophils, monocytes, and macrophages, and it has been found to be hypomethylated in hepatocellular carcinoma (HCC) tissues relative to adjacent tissues(Matmati et al., 2007; Archer et al., 2010). Peripheral blood EMR3 gene hypomethylation is associated with BC in China women(Zhou et al., 2021). The study of EMR3 in LUAD has not been reported yet, and we hypothesized that EMR3 can inhibit its expression in LUAD, thus leading to LUAD. WNT7A signaling can play a role in various types of tumors by initiating classical and non-classical pathways(Winn et al., 2006; Yoshioka et al., 2012). The canonical pathway (or Wnt/β-catenin) involves Wnt bind to Fzizzleds (Fzds) that acts as a negative regulator of β-catenin accumulation(MacDonald et al., 2009; Niehrs, 2012). WNT7A is closely related to a variety of tumors(Nusse et al., 1991), and which exerts tumor suppressive effects in a variety of cancers. Ochoa Hernandez AB demonstrated that the expression of WNT7A in normal peripheral blood T lymphocytes was significantly higher than that in leukemia cells(Ochoa-Hernández et al., 2012). Calvo R and Ohira T et al. found that WNT7A may play a tumor suppressor role in lung cancer (especially in NSCLC), and WNT7A is down-regulated in NSCLC(Calvo et al., 2000; Ohira et al., 2003). Ohira T et al. also found that WNT7A plays an important tumor suppression role in lung cancer, which may be related to the absence of E-cadherin(Hsieh et al., 1999). In our present study, WNT7A expression was lower in LUAD than in adjacent non-cancerous tissues, which were consistent with the above results.
The staging standard of LUAD in clinical medicine is mainly classified according to TNM staging standard that is accepted by the international. While, it lacks comprehensive evaluation based on gene expression quantity. In our study, we constructed a prognostic model based on risk score, T stage, and N stage. Moreover, we established a nomogram in the light of prognostic model. Nomogram incorporates assumed risk factors, calculates the proportion of each factor, and ultimately generates a numerical possibility for individual clinical outcomes(Iasonos et al., 2008). Nomogram has become a popular tool for tumor prognosis due to it intuitive visual presentation and personalized application. Through incorporating risk scores derived from autophagy-associated gene signatures, Liu et al. predicted the OS of 3- and 5- year in NSCLC via nomogram(Liu et al., 2019). Long and colleagues developed a nomogram that included TP53-associated immune prognostic features of hepatocellular carcinoma and other prognostic factors(Long et al., 2019). Consistently, our calibration curve further verified the clinical utilize of nomogram, indicating the accuracy and superior performance of the model prediction and it could well predict the 1- and 3- year survival probabilities for LUAD patients.
And meanwhile, there were 11 immune cells that differed remarkably between high/low risk groups, such as resting CD4 memory T cells, memory B cells, resting mast cells. The T-cell activation and activated T-cell (CD4 memory) pathways were predominantly involved in the immune microenvironment regulation of LUAD(Liu et al., 2021a). Meanwhile, the study of Wu Chunlei et al. also found the correlation between resting CD4 memory T cells and LUAD(Wu et al., 2021). Memory B cell was highly enriched in tumor tissues with better differentiation and increased frequencies of somatic hypermutation, it was reported that TP53 mutations diminish the immune response, resulting in reduced infiltration of memory B cell in LUAD patients(Hao et al., 2022; Song et al., 2022). In the study of Fenglong Bei et al., the higher content of resting mast cells indicated a better prognosis in Asians(Bie et al., 2021). However, in our study, the expression of resting mast cells in the low-risk group was lower than that in the high-risk group, which was not consistent with the finding of Chao Mao et al(Ma et al., 2021). Therefore, the expression of resting mast cells in LUAD patients and the specific mechanism need further study.
MiRNA is a class of small noncoding RNA (ncRNA) composed of 17-25 nucleotides(Wang et al., 2016). Especially in recent years, new achievements have been made in the study of miRNAs and human cancers(Alvarez-Garcia and Miska, 2005; Esquela-Kerscher and Slack, 2006). Simultaneously, TFs are proteins that bind to chromatin to activate or inhibit transcription by helical binding of DNA to specific regulatory sequences in the form of trans-activated or trans-repressive domains. Any changes in their expression may lead to uncontrolled cell integrity or dynamic homeostasis, leading to pathological changes(Lambert et al., 2018), such as diabetes(Polvani et al., 2016) and cancers(Pires et al., 2018). However, in-depth studies have found that TFs can play a double-edged sword role, so they may play both anticancer and oncogenic roles, even though TFs of the same family may have two-way effects on the same cancer(Rad et al., 2016; Song et al., 2016). Therefore, accurately understanding the role of miRNA-mRNA-TF in the LUAD is the focus of cancer mechanism and treatment researches in recent years. However, the specific regulatory mechanism targeting the miRNA-mRNA-TF network of LUAD in our study needs further exploration.
For five small molecule drugs targeting the risk model genes (ABCC2, BCL2L10, WNT7A), Tamoxifen is an estrogen receptor (ER) antagonist widely used in adjuvant chemotherapy for breast cancer(Phillips, 2001; White, 2003), and it has been found to induce apoptosis in colorectal, ovarian, prostate, and head and neck cancer cells(El Etreby et al., 2000; Tavassoli et al., 2002; Basu et al., 2004; Zhou et al., 2005). Moreover, Tamoxifen is also effective in treating ER-negative glioma tumors as a chemotherapeutic agent(Donson et al., 1999). Another recent study of Tamoxifen showed that it enhanced the cytotoxicity of erlotinib-induced human NSCLC cells by down-regulating the expression of Akt-mediated thymidine phosphorylase(Ko et al., 2014). One more example, Obatoclax is a synthetic derivative of bacterial prodiginines which can bind to all anti-apoptotic Bcl-2 family members and play a role in pan-Bcl-2 inhibitor(Kim and Lee, 2014). It has already been extensively tested in both solid malignancies and lymphoma and been used in early clinical trials(Campàs et al., 2006). The patients with NSCLC can tolerate Obatokla mesylate in combination with docetaxel(Chiappori et al., 2014). Synthetic estrogen diethylstilbestrol (DES) produces several reactive compounds by oxidative bioconversion. It proposes two mechanisms by which reactive metabolites may be involved in organotropic tumorigenesis of DES. The affinity of metabolites for estradiol receptors present in estrogen target organs is the first mechanism. This has been shown for epoxide alkenes of DES. Organ-specific oxidation of DES based on peroxidase is the second mechanism. It was found that the intermediates of the reaction bind to nucleic acids and proteins in the characteristic way of chemical carcinogens(Metzler and McLachlan, 1979). According to Yohan Seo et al., DES was identified as a selective ANO1 inhibitor through high-throughput screening. These results showed that ANO1 is an essential target in treating NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent(Seo et al., 2021). Therefore, the screening of NETs-related genes in our study, so as to predict gene-drug, the prediction results for the future treatment of LUAD have a better guiding role.
Through a series of bioinformatic analysis, the study constructed a prognostic risk model according to IGF2BP1, ABCC2, BCL2L10, FIGF, EMR3, and WNT7A in LUAD, providing new ideas for related studies of LUAD.
Despite the underlying clinical significance of our results, several limitations need to be considered. First, the clinical features extracted from the public databases are limited and incomplete. Potential prognostic factors (such as personal history, treatment, and background diseases) and environmental factors (including smoking, exposure to certain toxins, or treatments) affecting the risk model genes were unclear. Second, our risk model was constructed based on RNA-seq data. The procedure of sample treatment, RNA extraction, reverse transcription, and detection needs to be standardized. Third, although we have verified the expression of NRGs in LUAD, the specific regulatory mechanism is still unclear and needs further study. We will continue to study and pay more attention to the progress of NETs and LUAD.