ADRB2 expression predicts the clinical outcomes and is associated with immune cells infiltration in lung adenocarcinoma

The gene encoding beta2-adrenergic receptor (β2-AR), adrenoceptor beta 2 (ADRB2), has been reported to closely associated with various cancers. However, its role in lung adenocarcinoma (LUAD) remains controversial. This research shed light on the prognostic value of ADRB2 in LUAD and further explored its association with immune cell infiltration. ADRB2 was significantly decreased in LUAD. ADRB2 expression in LUAD was significantly correlated with gender, smoking status, T classification, and pathologic stage. Patients in the low ADRB2 expression group presented with significantly poorer overall survival (OS) and disease-specific survival (DSS). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) results showed that ADRB2 participates in immune response. The expression of ADRB2 was positively correlated with the infiltration level of most immune cells. Notably, ADRB2 is involved in LUAD progression partly by regulating the immune microenvironment, which may potentially serve as a significant prognostic biomarker as well as a potential drug target.

Zheng et al. 1 found that ADRB2 was underexpressed in LUAD tissues and low ADRB2 expression is associated with poor clinical outcomes. The other research by Wang et al. reached the same conclusion 10 .
Based on The Cancer Genome Atlas (TCGA) dataset, LUAD dataset was acquired for bioinformatics analysis to verify that ADRB2 expression was significantly down-regulated in LUAD. Next, the relationship between ADRB2 gene expression and clinical traits was further investigated. The expression of ADRB2 was highly correlated with immune infiltration, which further confirmed that ADRB2 could be used as a prognostic biomarker of LUAD.

Results
Patient characteristics. The clinical data of 535 LUAD patients included gender, age, smoking status, T stage, N stage, M stage, pathological stage, OS event, and DSS event (Table 1). Chi-square test revealed that ADRB2 was significantly correlated with T stage (P < 0.001), gender (P = 0.005), smoking (P = 0.004), OS events (P < 0.001) and DSS events (P = 0.005). There was no significant correlation between ADRB2 expression and other clinicopathological features.
ADRB2 expression level in LUAD. Based on the TCGA database, ADRB2 mRNA expression level was analyzed in 594 tissues. As showcased in Fig. 1a, it evaluated the ADRB2 mRNA expression levels in TCGA pancancer. The results indicated that the expression level of ADRB2 in the LUAD samples was much higher than that in the normal tissue samples (P < 0.001). Box plots showed ADRB2 mRNA expression levels in 59 adjacent non-tumor tissues and 535 LUAD tissues. As shown in Fig. 1b, ADRB2 was down-expression in LUAD tissues compared with those in normal tissues (P < 0.001, Fig. 1b). Figure 1c shows a pairwise boxplot of the same out- Table 1. Demographic and clinicopathological parameters of high and low ADRB2 expression group patients with lung adenocarcinoma in TCGA-LUAD.  Figure 1d shows 100 normal samples and 94 LUAD samples from the GEO database, which shows that ADRB2 is lowly expressed in LUAD patients (P < 0,001). ADRB2 expression in LUAD patients does not show correlation to age (Fig. 1e). Moreover, ADRB2 was significantly lower in males (P < 0.001, Fig. 1f) and in patients with a smoking history (P < 0.001, Fig. 1g).
Association between ADRB2 and TNM stages in LUAD patients. To better understand the impact of ADRB2 on LUAD patient prognosis, Kruskal-Wallis analysis and Spearman correlation analysis were performed to determine the relationship between ADRB2 expression and clinicopathological characteristics (pathologic and TNM stages). The ADRB2 expression was significantly decreased in LUAD patients, and was significantly correlated with pathological stage, T stage and N stage. (Fig. 2a-

Evaluation of the diagnostic capacity of ADRB2 in LUAD.
To explore the diagnostic value of ADRB2 for LUAD, receiver operating characteristic (ROC) curve analysis was performed. The results of the ROC curves indicated that ADRB2 was highly sensitive to the diagnosis of LUAD (AUC, 0.994; 95% CI 0.989-0.999, Fig. 5a). Additionally, the AUC was 0.598 for OS, which indicated that the prognostic model had good performance in predicting survival prognosis of patients with LUAD (AUC, 0.590; 95% CI 0.548-0.648, Fig. 5b). The timedependent accuracy of ADRB2 in predicting OS in 1, 3, and 5 years was also assessed through a time-dependent ROC analysis, AUC < 0.5 indicates that the expression of ADRB2 is opposite to the occurrence trend of OS events in LUAD patients (Fig. 5c). Figure 5d shows that the survival rate of patients in the high-risk group is poor, and the risk of death is high. With the decrease of ADRB2 expression, the risk score tend to increase gradually. Figure Fig. 6b,c). However, T gamma delta (Tgd) and type 2 Th cells (Th2) (P < 0.001, Fig. 6b,c) showed a negative association with ADRB2. As can be seen in ADRB2 associated gene set enrichment in LUAD. To determine ADRB2-related signaling pathways, GSEA was performed between the high-and low-ADRB2 groups. Significance was assessed using a normalized enrichment score (NES) ≥ 1.5, P ≤ 0.05, and false discovery rate (FDR) ≤ 0.25. KEGG pathway enrichment analysis indicated that 13 important signaling pathways were significantly enriched in the highly expressed ADRB2 phenotypes, including the JAK STAT signaling pathways, leukocyte trans-endothelial migration, chemokine signaling pathway, autoimmune, thyroid disease, Fc epsilon ri signaling pathway, intestinal immune network for iga production, cytokine receptor interaction, B cell receptor signaling pathway, NK cell-mediated cytotoxicity, allograft rejection, Mapk signaling pathway, T cell receptor signaling pathway, and NSCLC. Meanwhile, there were 13 eligible signaling pathways enriched in the low-ADRB2 expression, including spliceosome, RNA polymerase, RNA degradation, citrate cycle (or TCA cycle), cell cycle, pentose phosphate pathway, basal transcription factors, oxidative phosphorylation, DNA replication, mismatch repair, cysteine and methionine metabolism, ubiquitin-mediated-proteolysis, and amino sugar and nucleotide sugar metabolism (Table 3, Fig. 7). These results contribute to further exploration of ADRB2 pathophysiological mechanisms.

Discussion
LUAD is a type of malignant lung tumor that originates from the bronchial mucosal glandular epithelium. LUAD is characterized by inconspicuous early symptoms, and LUAD is a lung tumor with a significant rate of malignant recurrence, metastasis, and unsatisfactory prognosis. Early diagnosis is difficult in most patients. ADRB2 is ubiquitously expressed in multiple tissues, including the smooth muscle of the human bronchi, cardiovascular system, central nervous system, and gastrointestinal tract. In recent years, increasing evidence shows that ADRB2 has a vital place in the occurrence and development of diverse range of cancers. Zhang et al. 11 found that the mRNA expressions of ADRB2 were higher in gastric cancers compared with normal tissues. Moreover, patients with gastric cancer with positive ADRB2 expression exhibited larger tumor size, late clinical stage, lower differentiation, and distant metastasis. In addition, high ADRB2 expression can promote the angiogenic switch in prostate cancer and prevent or delay the dominant role of pro-angiogenic factors, leading to tumor progression 12 . β2-AR is encoded by ADRB2 and can bind specifically to endogenous catecholamines (such as adrenaline and noradrenaline), and promotes the production and release of cyclic adenosine phosphate (cAMP). cAMP can further activate and phosphorylate protein kinase A and C to activate downstream signal transduction pathways and promote the proliferation, migration, and metastasis of lung cancer cells 13 . The positive ADRB2 expression can occur in several cancers, including hepatocellular carcinoma, colorectal cancer, melanoma, and gastric cancer,  17 . In oral squamous cell carcinoma, patients with higher ADRB2 had a significant longer DSS and OS 18 . Caparica et al. 19 found that a high ADRB2 expression may be a favorable prognostic factor in patients with HER2 + early breast cancer and evidence that a possible association with antiproliferative, antiangiogenic, and immunogenic effects of ADRB2. Yazawa et al. 20 retrospectively analyzed 328 surgically-resected patients with NSCLC and found that positive ADRB2 expression was found in 29% of LUAD tissues, which markedly increased compared with in non-adenocarcinoma tissues. A high level of ADRB2 expression was associated with vascular invasion, tumor cell proliferation, and poor prognosis in patients with LUAD. Nevertheless, Wang et al. 10 searched the gene expression synthesis (GEO) to obtain data showing that ADRB2 is down-regulated in LUAD, and ADRB2 mRNA levels declined with stage progression. ADRB2 mRNA expression levels and its gene product, β2-AR, differ. The exact mechanism is still controversial. This study used high-throughput RNA sequencing data obtained from TCGA database to further ascertain the expression and the prognostic significance of ADRB2 in LUAD and explore its correlation with immune cell infiltration. The low expression of ADRB2 in tumor tissues of LUAD patients is correlated with gender, smoking, OS events and DSS events, indicating that ADRB2 is a potential biomarker for prognosis and diagnosis. Data mining from TCGA database showed that the expression of ADRB2 was correlated with pathological stage, T stage and N stage, and the disease tended to progress with the decrease of ADRB2 expression. Premised on this, logistic regression analysis showed that ADRB2 is significantly correlated with gender, smoking status, T stage and pathological stage. The survival analysis revealed a favorable survival in high ADRB2 expression group compared to those with low expression. Univariate analysis revealed that pathological stage, T stage, N stage, M stage, and ADRB2 expression influenced OS. To further determine the diagnostic capacity of ADRB2 in LUAD, ROC curves were used to confirm that ADRB2 is sensitive to the diagnosis and prognosis of LUAD. Altogether, these findings illustrate that ADRB2 is a potential prognostic biomarker for LUAD.
The tumor immune microenvironment (TME) is very important in cancer pathogenesis 21 . Immune cells are vital elements of the TME 22 . The correlation between ADRB2 expression and the infiltration of 24 immunocytes was further explored to elucidate the mechanisms responsible for ADRB2 to predict clinical prognosis. Further correlation analysis indicated that the infiltration of 19 immune cells was significantly associated with ADRB2 expression. ADRB2 expression was positively correlated with aDCs, B cells, CD8 + T cells, cytotoxic cells, DCs, eosinophils, iDCs, macrophages, mast cells, neutrophils, NK cells, pDCs, T cells, T helper cells, Tcm, Tem, TFH, Th1 cells, Th17 cells, and TReg. B cells are dominant in the progression of lung cancers 23,24 and can be observed at the individual stages of carcinogenesis 25 . B cells can prolong the survival of cancer patients by inhibiting tumor progression and preventing metastasis. In addition, antibodies produced by B cells are essential mediators of tumor cell death 26 . Cytotoxic T lymphocytes (CTLs) are major players in antitumor immunity and can lead to www.nature.com/scientificreports/ apoptosis of cancer cells through a series of steps; therefore, high infiltration of CTLs is a favorable prognostic marker for many cancers. DCs are the most effective antigen-presenting cells to induce primary tumor immune response 27 , and in NSCLC patients, an increased DC count was significantly associated with an increase in DSS 28 . The role of macrophages in cancer progression is still controversial. Tumor-associated macrophages promote tumor progression by facilitating tumor stroma formation and angiogenesis 29 . In patients with NSCLC with prolonged survival, macrophage-infiltrating tumors are mainly of the M1 type 30 . Mast cells, which have cytotoxic effects on cancer cells, can enhance the immunity of patients with LUAD against cancer cells and improve their postoperative prognosis 31 . NK cells are cytotoxic and it is essential in the immune monitoring of cancers 32 . Carrega et al. 33 found that in resected LUAD tissues, increasing numbers of infiltrating NK cells were associated with favorable patient survival outcomes. T cells are the most abundant monocytes infiltrating the NSCLCs 34 . T cells can secrete cytokines to inhibit tumor stroma formation and use cytotoxic molecules to kill epithelial nuclear stromal cells. Al-Shibli et al. 35 reported that T cell infiltration is associated with better DSS, and T cells are an independent indicator of survival. T helper cells play an important role in cancer immunity by secreting cytokines 36 . Both Th1 and Th17 cells produce proinflammatory factors, and their extensive infiltration can significantly improve clinical outcomes in a variety of cancers 37,38 . Large infiltration of cytotoxic T cells in tumor tissues is associated with longer survival 35 . Studies have shown that TFH has an antitumor response, and IL-21 secreted by TFH induces the activation, proliferation, and differentiation of B cells 39,40 . ADRB2 expression may up-regulate the levels of infiltrating immune cells to limit the development of LUAD. In contrast, ADRB2 expression was negatively correlated with Th2 cells and Tgd. Th2 cells have many pro-neoplastic activities and take part in cancer progression by cytokine release. Th2 cells are dominant in lymphocytes from malignant pleural effusion in patients with lung cancer 41,42 . Current studies have revealed that Tgd has a pro-tumor effect, which can inhibit innate and adaptive immunity by inducing immunosenescence [43][44][45] . To summarize, low ADRB2 expression is associated with poor prognosis of LUAD. These results show that ADRB2 expression level affects the immunity activity in the TME, and ADRB2 might be a valuable biomarker for the immune status in LUAD patients.
To further explore the mechanism of ADRB2 in LUAD, the signaling pathways involved in ADRB2 was screened. In the ADRB2 high-expression group, ADRB2 associated genes were significantly enriched in immune signaling pathways (such as B cell receptor signaling pathway, T cell receptor signaling pathway, and NSCLC, NKcell-mediated cytotoxicity, chemokine signaling pathway, and Jak STAT signaling pathway), in KEGG analysis. Those are significant in the tumorigenesis, development, and invasion of malignancies 46 . On the other hand, in the ADRB2 low-expression group, ADRB2 correlated genes were enriched in metabolism-related pathways, including RNA polymerase, citrate cycle, pentose phosphate pathway, oxidative phosphorylation, cysteine and methionine metabolism, and amino sugar and nucleotide sugar metabolism, implying that ADRB2 up-regulated the signaling pathways associated with immune response and induced antitumor efficiency. Therefore, ADRB2 expression was down-regulated as LUAD progressed, and the TME switched from an immune-active state to a metabolic state. The ADRB2 expression can be considered a biomarker to predict immune response.
At present, most studies on the relationship between ADRB2 and the occurrence and progression of LUAD are based on its gene expression product, β2-AR, and its signaling pathway. This study revealed ADRB2 as a key gene in the immune microenvironment of LUAD by performing a bioinformatics analysis, to provide evidence for ADRB2 as a potential prognostic marker for LUAD. However, some limitations arise in the research. Firstly, the present research was limited by the small number of cases, and a large cohort is needed to validate the results of this research. Secondly, this research primarily focused on the expression of ADRB2 mRNA from TCGA, and without involving β2-AR levels in LUAD tissues. Thus, this study still needs large-sample, multicenter, multi-ethnic clinical trials and basic experimental studies to prove the prognostic value of ADRB2 in LUAD. Low expression of ADRB2 is associated with poor prognosis in LUAD patients, which may be related to immunocompromised.

Conclusion
In summary, ADRB2 expression was significantly down-regulated in patients with LUAD. ADRB2 is involved in LUAD progression partly by regulating the immune microenvironment, which may potentially serve as a significant prognostic biomarker as well as a potential drug target.

Materials and methods
Data acquisition. On or before November 13, 2021, the mRNA profile (HTSeq-Counts and HTSeq-FPKM) was extracted from TCGA (https:// cance rgeno me. nih. gov/), including 535 LUAD samples and 59 normal samples. Relevant clinical information was derived from TCGA. RNAseq data in FPKM (Fregments Per Kilobase Per Million) format were converted to TPM (transcripts Per Million reads) format and log2 transformed. ADRB2 expression data from datasets GSE40791 was downloaded from the GEO database (https:// www. ncbi. nlm. nih. gov/ gds/). The relevant data TCGA and GEO provided is open-access, no additional approval from the Ethics Committee were required. All methods were performed in accordance with the relevant guidelines and regulations.
ADRB2 expression and survival analyses. The original expression data downloaded from TCGA were processed using the Perl programming language (version: strawberry-perl-5. 32 Gene set enrichment analysis (GSEA). All LUAD patients in TCGA dataset were allocated into high and low group based on the expression of ADRB2. GSEA was used as a signaling pathway analysis tool to explore the signaling pathways related to ADRB2 in LUAD. GSEA between high and low ADRB2 expression was performed using GSEA 3.0 (http:// www. gsea-msigdb. org/ gsea/ index. jsp). Phenotypes were determined based on ADRB2 expression levels. The gene set "c2 all.v6.0 symbols.gmt" was used for the enrichment analysis. KEGG analysis was performed to explore the significant pathways associated with ADRB2 expression.
Statistical analysis. All statistical analyses were performed using IBM SPSS Statistics for Windows, version 23.0 (IBM Corp, Armonk, NY, USA) and R (version 4.0.2, https:// www.r-proje ct. org/) and the level of statistical significance was defined as a P < 0.05.