Targeting the ARNTL2 Gene as a Potential Strategy for Lung Adenocarcinoma

The relationship between biorhythm and cancer has been increasingly reported in recent years. As one of the core transcription factors of biorhythm, ARNTL2 is thought to be implicated in the occurrence and development of many malignant tumors, such as breast cancer. However, the role of ARNTL2 in lung adenocarcinoma remains elusive. In the current study, we found that expression of ARNTL2 was markedly upregulated in lung adenocarcinoma, and high ARNTL2 expression was correlated with advanced N stage and poor survival in patients. Moreover, ARNTL2 expression levels are closely associated with many important features of lung adenocarcinoma, such as tumor immune microenvironment, ferroptosis, microsatellite instability, tumor mutational load, and drug sensitivity. ARNTL2 could also promote proliferation, invasion, and metastasis of lung adenocarcinoma cells. In addition, we found that high ARNTL2 expression might promote epithelial-mesenchymal transition, metastasis and promotion of angiogenesis of tumor cells. In conclusion, our study reveals that ARNTL2 is a prognostic and promising therapeutic biomarker for people with lung adenocarcinoma. assay


Introduction
Lung cancer is the foremost cause of cancer death, and it has the second-highest incidence worldwide in 2020 1 . Among the primary lung cancers, lung adenocarcinoma is the most common subtype 2 . Although efforts and improvements have been made in therapy for lung adenocarcinoma (LUAD), the prognosis of LUAD patients is still not optimistic, the 5-year survival rate of lung cancer patients was less than 20% 3 .
Thus, it is of great importance to determine the etiology and mechanisms of LUAD malignant progression and explore more effective treatment strategies.
Circadian rhythms affect many physiological processes, it is widely disrupted in cancers, and its role in the tumor has also been revealed by increasing studies in recent years. Aryl Hydrocarbon Receptor Nuclear Translocator Like 2 (ARNTL2), as a critical circadian transcription factor 4,5 , has been shown to play an important role in a variety of tumors, such as colorectal cancer, kidney cancer, and pancreatic ductal adenocarcinoma. However, studies on the role of ARNTL2 in lung adenocarcinoma are relatively rare and not su ciently thorough.
The current study comprehensively explored the relationship between the ARNTL2 and lung adenocarcinoma, in order to explore the roles of ARNTL2 in the development, progression and treatment of lung adenocarcinoma. The effects of ARNTL2 on human lung adenocarcinoma cells in terms of tumor mutational burden (TMB), microsatellite instability (MSI), immune microenvironment, ferroptosis, and drug sensitivity were further evaluated, aiming to provide potential targets and ideas for the treatment of lung adenocarcinoma. In addition, we experimentally demonstrated that ARNTL2 promotes tumor cell proliferation, migration, and invasion in lung adenocarcinoma cell lines A549 and H1299.

Result
Gene expression with clinical features A previous study found that ARNTL2 was frequently highly expressed in many cancers 6 . The differential expression between tumor and adjacent normal tissues for ARNTL2 across TCGA is shown in Figure 1A. To further validate whether ARNTL2 was differentially expressed in lung adenocarcinoma tissues, we conducted an analysis of 513 cases of LUAD patient data from the TCGA database, which showed signi cantly higher ARNTL2 expression in LUAD tissues than in normal adjacent tissues, (P < 0.001). The same conclusion was obtained from the validation after combining the GTEx data ( Figure 1B). In addition, we divided the lung adenocarcinoma patients in the TCGA database equally into two groups according to the high and low expression of ARNTL2, and found there were differences in the baseline characteristics of the two groups (Table 1 and Supplementary Figure 1). Speci cally, patients in the high-ARNTL2 group were more male and had more advanced N stage and AJCC stage.
For lung adenocarcinoma patients in the TCGA database, we also validated the correlation between survival and clinical factors. A total of 513 lung adenocarcinoma participants were involved in univariate and multivariate analyses to validate survival factors. Univariate analyses revealed that ARNTL2 (p 0.001), T stage (p 0.001), and N stage (p 0.001) were statistically signi cant predictors of tumorspeci c survival. According to multivariate analysis, ARNTL2 (p 0.001), T stage (p = 0.005), and N stage (p = 0.001) remained independent prognostic predictors for LUAD patients. The details of the correlations between survival outcomes and parameters are shown in the following forest plot ( Figure 1D).

TMB and mRNAsi analyses
As one of the immunotherapy biomarkers, tumor mutation burden (TMB) has attracted more and more attention in recent years, we analyzed the relationship between ARNTL2 gene expression and TMB in lung adenocarcinoma patients according to the TCGA database, and the results showed that there was a degree of positive correlation between ARNTL2 gene expression and TMB (Figure 2A), which may indicate that in lung adenocarcinoma patients, as ARNTL2 gene expression increases, TMB also increases. Thus ARNTL2 may have a reference value for immunotherapy.
It has been previously shown that mRNAsi is a valid method to evaluate the level of tumor differentiation.
Histopathology con rmed that higher values of mRNAsi tend to represent a greater degree of tumor dedifferentiation. AS shown in Figure 2B, in lung adenocarcinoma, the mRNAsi was signi cantly higher in the lung tumor specimens than in normal lung tissue. Furthermore, compared with the low ARNTL2 expression group, the high ARNTL2 group showed signi cantly higher mRNAsi, which may be related to the worse prognosis of patients in the high ARNTL2 group.

MSI
Microsatellites (MS), also known as short tandem repeats (STR) or simple sequence repeats (SSR), are structures consisting of repetitive sequences of 1-6 nucleotides 7 . MSI is an important factor in tumorigenesis and progression and has been extensively studied in tumors such as colorectal cancer.
Early diagnosis of MSI is of great signi cance to the prognosis and treatment of MSI 8 . We found that the expression level of ARNTL2 and MSI were negatively correlated in lung adenocarcinoma patients ( Figure  2C).

Somatic mutations
Based on previous reports, the number of somatic mutations is thought to correlate with prognosis. The changes in the copy numbers' variation and distribution of somatic mutations in high-ARNTL2 and low-ARNTL2 groups were analyzed. The results showed that TP53 had a high mutation rate in the high-ARNTL2 group (55% vs. 41%, P-value < 0.01). In contrast, some genes, such as RELN (11% for the low subgroup and 18% for the high subgroup), had a lower mutation rate in the high ARNTL2 group ( Figure   2D).

DEG, GO, KEGG analyses and single-cell analysis
The prognosis of lung adenocarcinoma patients with different ARNTL2 expression levels may be related to DEGs. So, we have analyzed the expression pro les from the TCGA data, to derive the landscape of biological differences. The prognosis of lung adenocarcinoma patients with different ARNTL2 expression levels may be related to DEGs. So, we have analyzed the expression of DEGs to derive landscape of biological differences ( Figure 3A), and 185 signi cant different genes were found, including 114 upregulated genes in the high-ARNTL2 group such as SLC2A1, CD109, and ADGRF4 (adjusted P < 0.01), and 71 downregulated genes including C16orf89, IRX5, and IRX3 (Supplementary Table 2).
Functional enrichment of GO and KEGG in the 185 DEGs were analyzed. These pathways were associated with high-ARNTL2 expression. Cell cycle, apoptosis, IL-17 signaling pathway, and p53 signaling pathway ranked top in the high-ARNTL2 group ( Figure 3B).
In addition, we performed a single-cell analysis of lung adenocarcinoma cells through the CancerSEA database. The results showed that ARNTL2 gene expression was highly correlated with Angiogenesis, EMT, and Metastasis ( Figure 3C, Supplementary Table 3).

Immune in ltration and immune checkpoint analyses
The composition and proportion of immune cells in the tumor microenvironment (TME) have a signi cant impact on tumor development and treatment 9 . In lung adenocarcinoma, CD8+ and CD4+ T cells showed a signi cant positive correlation with ARNTL2 expression. In contrast, NK cell and Macrophage M2 showed a negative correlation ( Figure 4A). In addition, some common genes closely related to immune checkpoints also differed with the high expression of ARNTL2 ( Figure 4B). Compared to the low-ARNTL2 expression group, the high expression group had signi cantly higher expression of CD274, CTLA4, LAG3, and PDCD1, etc. These differences in the immune microenvironment may provide ideas for immunotherapy.

Ferroptosis and drug-sensitive analyses
Comparison of important genes associated with ferroptosis between the high and low expression ARNTL2 groups showed that ACSL4, CDKN1A, and MT1G, etc. were overexpressed in the high expression group ( Figure 4C), which may predict a correlation between ARNTL2 expression and ferroptosis in lung adenocarcinoma.
ANRTL2 expression also appears to have effects on drug sensitivity in lung adenocarcinoma, with IC50s signi cantly lower in the high ANRTL2 expression group than in the low ANRTL2 expression group for Ge tinib, Gemcitabine, Cisplatin, and Paclitaxel, although there was no signi cant difference in sensitivity to erlotinib between the two groups ( Figure 4D).
IHC and immuno uorescence: ARNTL2 is highly expressed in lung adenocarcinoma tissues The results of immunohistochemistry further con rmed the increased expression of ARNTL2 in lung adenocarcinoma. As shown in Figure 5A, the overexpression of ARNTL2 was seen in resected tumor tissues compared with adjacent normal tissues. Similar results were obtained by immuno uorescence analysis ( Figure 5B).

ARNTL2 promotes proliferation, invasion, and migration of LUAD cells
To identify the effect of ARNTL2 on proliferation and migration of lung adenocarcinoma cells, we established cell model of ARNTL2 downregulation by stably transducing with ARNTL2 shRNA-expressing lentiviruses in A549 and H1299 cells. Next, we detected the expression level of ARNTL2 by Western blot after transfection, the stable knockdown e ciency of shRNA at protein expression level was veri ed ( Figure 5C). ARNTL2 knockdown was very e cient in A549 and H1299 cell lines We subsequently performed CCK8, Clone formation, wound healing, and transwell assays to explore the effect of ARNTL2 in tumor proliferation and migration. CCK8 assays showed when ARNTL2 was downregulated, compared with the control cells, the proliferation ability was decreased in both A549 and H1299 cells ( Figure 5D). To further testify the role of ARNTL2 in lung adenocarcinoma formation, Clone formation of ARNTL2-knockdown and control cells was assessed. The results showed that ARNTL2knockdown cells formed signi cantly fewer colonies compared to control cells ( Figure 5E), which indicates ARNTL2 is essential for the oncogenicity of lung adenocarcinoma cells.
We also investigated whether ARNTL2-knockdown cells affected migratory and invasion. ARNTL2knockdown signi cantly reduced the migration of A549 and H1299 cells in a wound-healing assay, shControl cells almost recovered the wound within 48 hours, but shARNTL2 cells still have large areas of unhealed wounds ( Figure 6A). As shown in gure 6B, ARNTL2-knockdown cells also showed signi cantly lower in ltration rates in the transwell migration and invasion assays compared to control cells (p < 0.05).
These results suggest that ARNTL2 may be a key promoter of lung adenocarcinoma cell growth and invasion.

Discussion
Circadian rhythm disorders may be associated with a variety of diseases, such as obesity and depression 10,11 . In addition, Strong epidemiological evidence links circadian disruption with cancers 12 . Moreover, in recent years, a large number of studies have demonstrated that circadian rhythm disorders may be associated with the development of various cancers in human being 13,14 . As an important biorhythm-related gene, ARNTL has been reported to play an oncogenic role in many human cancers. For example, Prior studies found that upregulation of ARNTL2 is associated with poor survival and immune in ltration in clear cell renal cell carcinoma 15  In this study, we performed a comprehensive analysis by integrating available data from TCGA and GTEx database. We found that ARNTL2 expression was correlated with the pathological N-stage of lung adenocarcinoma patients. Similar results have been reported in colorectal cancer. And patients with high ARNTL2 expression had a worse prognosis and could serve as an independent predictor of prognosis in lung adenocarcinoma. Furthermore, a prognostic nomogram including ARNTL2 based upon the results of multivariate Cox analysis was constructed to predict the long-term survival of LUAD patients. Our study also con rmed that ARNTL2 is upregulated in lung adenocarcinoma, which is consistent with some previous reports: Arntl2 was upregulated in BLCA, BRCA, COAD, and READ 18 . ARNTL2 can also promote proliferation and migration of lung adenocarcinoma cells, which suggests that ARNTL2 may be a key promoter of lung adenocarcinoma cell growth and invasion. In addition, a positive correlation between ARNTL2 expression and TMB was found in lung adenocarcinoma. In contrast, MSI showed a negative correlation with ARNTL2 expression.
Although much progress in the treatment of lung cancer has been made in recent decades, lung cancer remains the deadliest malignancy worldwide and signi cantly affects the quality of life of patients 19 .
Especially for advanced patients, the 5-year survival rate of stage IV lung cancer patients is only 4.7% 20 . Immunotherapy is currently attracting increasing attention in the treatment of lung cancer. However, there are still many patients who bene t less from immunotherapy due to insensitivity to drugs. Therefore, it is vital to discover new effective prognostic biomarkers and speci c immune-related therapeutic targets.
Our study shows that in lung adenocarcinoma, ARNTL2 causes a signi cant impact on the immune microenvironment.  [21][22][23] . Therefore, a previous study suggested that immunotherapy targeting NK cells may be a breakthrough point in treatment for lung cancer 24 . There were also signi cant differences in immune checkpoints between high-ARNTL2, and low-ARNTL2 group patients. Immunecheckpoint inhibitors (ICIs) is currently making a splash in the treatment of non-small cell lung cancer 25,26 , further research on the relationship between ARNTL2 and immune checkpoints may be meaningful and necessary. In addition, numerous studies have demonstrated that TMB and MSI can be used to predict the effect of immunotherapy 27, 28 , for example, tumor cells with high TMB are reported to be more easily recognized by immune cells, and thus respond more signi cantly to immunotherapy 29 , which may be used in combination with TNM staging to predict the progression and prognosis of patients with advanced tumors. We found that in lung adenocarcinoma, tumor immune in ltration, immune checkpoints, MSI and TMB were all associated with ARNTL2 expression levels. The above ndings may deserve more in-depth studies and help with immunotherapy for a subset of lung adenocarcinoma patients in the future.
We found that ARNTL2 gene expression was highly correlated with EMT and Metastasis by single-cell analysis, which This could potentially explain why LUAD patients with high-ARNTL2 expression have a worse prognosis. Indeed, the role of ARNTL2 in promoting tumor invasion and metastasis has been reported in a variety of tumors. A previous study of colorectal cancer found that high ARNTL2 expression was signi cantly associated with vascular invasion and lymph node metastasis 16 . In addition, several studies have shown that ARNTL2 is associated with the development and metastasis of breast cancer 30 , and Ha NH, et al. indicated that affect the expression levels of ARNTL2 has a signi cant effect on metastatic progression in ER-breast cancers 31 . It has also been demonstrated that in PDAC, ARNTL2 could affect tumor proliferation, migration, and invasion through the TGF/BETA pathway 6 . Brady et al.
reported that ARNTL2 could drive metastatic self-su ciency in lung cancer 32 . Moreover, Epithelial-to-mesenchymal transition (EMT) has been increasingly recognized to promote carcinoma invasion and metastasis. A previous study con rmed that in colon carcinoma, downregulation of ARNTL2 could suppress tumor cell proliferation and migration via SMOC2-EMT through inactivation of PI3K/AKT signaling pathway 33 . These studies suggest that ARNTL2 may contribute to tumor development through multiple mechanisms, such as the promotion of metastasis and EMT.
By applying bioinformatics analysis, we found that there was a high correlation between ARNTL2 expression level and promotional effect on angiogenesis in lung adenocarcinoma cells. In addition, ARNTL2 expression levels appear to have an effect on ferroptosis and drug sensitivity in lung adenocarcinoma cells. However, there are no reports on related elds, further experimental validation is required. This will be the next step of our future work which may could help to reveal deeper mechanisms.
There were also some limitations to this study. Although this study used a massive cohort of TCGA and GEO databases to develop and validate the role of ARNTL2 in lung adenocarcinoma, selection bias could not be avoided because of the retrospective nature of our study design. In addition, this experiment did not conduct more in-depth experiments to explore the mechanism of ARNTL promoting tumor metastasis and EMT in lung adenocarcinoma, which is also the direction of our future research.
ARNTL2 is highly expressed in lung adenocarcinoma, and high ARNTL2 expression was associated with lymph node metastasis and was an independent predictor of worse prognosis for lung adenocarcinoma patients. Notably, the expression of ARNTL2 in lung adenocarcinoma is associated with a variety of features such as tumor immunity, ferroptosis, MSI, and drug sensitivity, which may provide a prediction and reference for immunotherapy. ARNTL2 may also be associated with the promotion of tumor metastasis, EMT, and angiogenesis. Through experiments, we veri ed that it could promote the proliferation, invasion and metastasis of lung adenocarcinoma. In conclusion, ARNTL2 may be a potential biomarker for lung adenocarcinoma, it may be useful to predict prognosis and guide the treatment in a subgroup of LUAD patients, and may be a new target for therapeutic approaches. TCGA cases were divided into high or low groups based on the median values of ARNTL2 expression level. Genome statistical analyses performed in R version 4.0.3 were as follows:

Data processing
(a) differentially expressed genes (DEGs): The limma package was used to identify DEGs and miRNAs. The moderated t-test was adopted to calculate DEGs and miRNA expression changes, and the P value was adjusted as FDR by Benjamini and Hochberg method. The log fold change > 0.5, and the adjusted P value < 0.05 35 .
(b) copy number variations (CNV) and microRNAs (miRNAs): the maftools package were used to compare the distribution of somatic mutations and the types of copy number variations 36 . The adjusted P value < 0.01 was used to assess the signi cance of the mutational frequency. The somatic mutations and the types of copy number variations between high and low-ARNTL2 groups were compared by Kruskal-Wallis test, and the adjusted p-value <0.05 were considered statistically signi cant. Results were shown with the oncoplot function.
(c) GO and KEGG: The involved pathways and biological functions of the DEGs were performed with GO and KEGG pathway enrichment analysis by the "clusterPro ler" R package 35 . The cut-off value was set as adjusted P < 0.05 and false discovery rate (FDR) < 0.05. (h) Correlation analysis of ARNTL2 expression and TMB/MSI 44, 45 : we used Spearman's correlation analysis to describe the correlation between quantitative variables without a normal distribution. The value range of Pearson correlation coe cient is [-1,1] with a higher absolute value indicating a stronger association and the sign indicating a positive or negative association between the two variables. The density curve on the right represents the distribution trend of the TMB/MSI score; the upper density curve represents the distribution trend of the gene.
All the above analysis methods and R package were implemented by R foundation for statistical computing (2020) version 4.0.3.

Single-cell analysis
Single-cell analysis was performed through the CANCERSEA website. CancerSEA is the rst dedicated database that aims to comprehensively decode distinct functional states of cancer cells at single-cell resolution 46 .

Prognosis analysis
Prognostic information was analyzed in the PrognoScan database and Kaplan-Meier plotter database 47 .

Statistical analysis
Statistical analyses in the current study were completed by R version 3.6.1 (R Foundation for Statistical Computing, Vienna, Austria). The R package included survival, rms and ggplot2. Statistical signi cance was set at a two-sided p value < 0.05. Categorical variables were compared using Fisher's exact test and Pearson's c2 test and continuous variables were compared using Student's t-test and the Wilcoxon test.
Multivariable Cox regression analyses were used to test independent prognostic value using the R package survival and the coxph function.

Cell culture and lentivirus infection
Lung adenocarcinoma cell lines (A549 and H1299) were purchased from the Chinese Academy of Sciences Cell Bank. Cells were cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin, and 100 U/mL streptomycin in a humidi ed 5% CO2 atmosphere at 37°C.
Two different short hairpin RNAs (shRNAs) encoding shARNTL2 and scramble shRNA control (catalogue) were designed by Shanghai Genechem Co.,Ltd. and cloned into a lentivirus vector with puromycin resistance, which was then transfected into cells and screened with puromycin to ensure transfection e cacy. Viral transduction was performed as the manufacturer's protocol. After 3 days of virus transfection, knockdown was veri ed by western blot analysis using ARNTL2-speci c antibody (ab221557, abcam). Sequences of the shRNAs and the control are provided in Supplementary table 1.
Western blot RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors cocktail (Beyotime) were used to extract proteins, enhanced BCA Protein Assay Kit (Beyotime) was used for protein quanti cation. As previously reported, Protein samples were separated by electrophoresis on SDS-PAGE and transferred to a polyvinylidene di uoride membrane (Merck-Millipore, Burlington, MA, USA).
After the transfer, the blots were blocked with 5% milk for 1 hour and incubated with primary antibody for 12h at 4°C. Then, tris-buffered saline Tween-20 (TBST) was used to wash the membranes three times.
After washing, the membranes were incubated with secondary antibodies at room temperature for 1 h.
Finally, the protein bands were visualized and analyzed using a Moon Chemiluminescence Reagent kit (Beyotime). The following antibodies were used in this study: Anti-ANRTL2 antibody (

Wound healing assay
Control shRNA and shARNTL2 cells were inoculated on the 6-well plate on average. Cells were grown into monolayer and manual scratching with a 200-µl pipette tip, then cells were rinsed with PBS and incubated at 37°C in serum-free media. Photographs of the wounded areas were taken every 24h by phase-contrast microscopy.

Transwell migration and invasion assay
Cell migration and invasion abilities were measured by Transwell assays using the 24-well transwell chambers with 8 µm polycarbonate membranes (Corning, NY). the uncoated were used to determine migration and pre-coated with Matrigel Basement Membrane Matrix were used to determine invasion (BD Biosciences). The chambers were rehydrated in a serum-free medium for 2 hours as described by the manufacturer. Then the upper chambers were added with serum-free medium, while the lower chambers were added with serum medium. Cells were seeded onto the upper chambers with a density of 5×104 cells per well, and incubated for 24 h at 37°C, 5% CO2. Cells migrated toward the lower chambers were xed with methanol and stained with 0.5% crystal violet. Each assay was photographed under the inverted microscope (Olympus), and the number of cells within each chamber was counted by ImageJ software.

Clone formation assay
Control or ARNTL2 shRNA-transduced A549 and H1299 cells (3 × 103 cells/well) were cultured in 6-well plates were cultured at 37°C in 5% CO2 environment. ARNTL2 shRNA-transduced or control cells were seeded in 6-well plates at a density of 5 × 106 cells per well and cultured at 37°C in 5% CO2 environment. After 9 days, cells were stained with 4% formaldehyde/0.005% gentian violet solution and captured under the inverted microscope.

Immunohistochemistry and immuno uorescence
The tissue specimens were collected from both tumor and adjacent normal tissues of 100 patients with LUAD who received surgery from September to November 2020 in the Zhongshan Hospital. As previously reported 48 , the tissues were stained by a GTVision + Detection System/Mo&Rb Immunohistochemistry kit (GK500710, GeneTech, Shanghai, China) following the manufacturer's protocol. Speci cally, the 5-µm para n-embedded tissues were dewaxed, rehydrated, and incubated with antibodies against ARNTL2 (1:500, Abcam, Cambridge, UK) at 4°C overnight, and then were incubated with biotinylated secondary antibodies. For immuno uorescence, sections were incubated with primary antibodies against ARNTL2 (rabbit polyclonal, 1:500), followed by incubation with the respective secondary antibodies (Cy3-labeled goat anti-rabbit IgG). DAPI nuclear counterstaining was then performed. Finally, uorescence microscope was used to take micrographs.      A Comparison of Wound-healing assays between ARNTL2-knockdown group and control group: ARNTL2 knockdown decreases cell migration in LUAD cells compared to control cells. B The invasion and migration capability of LUAD cells transfected with NC or sh-ARNTL2 was analyzed by transwell assay: ARNTL2 knockdown diminished migration and invasion ability of LUAD cells.