Methylation-regulated tumor suppressor gene HOXB4 inhibits human lung adenocarcinoma A549 cell invasion and metastasis through the RAP1 signaling pathway

Introduction

Lung cancer remains the leading cause of cancer morbidity and mortality, with almost 2.5 million new cases and over 1.8 million deaths worldwide in 2022 (1). There are various types of lung cancers, such as non-small cell lung cancer (NSCLC) which accounts for approximately 85% of all lung cancers. Lung adenocarcinoma (LUAD) is the most common histological subtype of NSCLC, accounting for approximately 40–45% of all cases (2,3). Currently, LUAD treatments mainly include surgical resection, chemotherapy, radiation therapy and immunotherapy. Despite advances in these treatments, the prognoses of patients with LUAD remain unsatisfactory, mainly owing to cancer progression and metastasis, as well as the lack of effective therapeutic targets (4,5). Therefore, it is urgent to identify effective biomarkers and therapeutic targets for LUAD.

The homeobox (HOX) genes, a subgroup of transcription factors containing a highly conserved homeodomain, play pivotal roles in stem cell self-renewal and tumorigenesis (6). Homeobox B4 (HOXB4) is a positive regulator involved in the self-renewal of hematopoietic stem cells (7) and is considered to be either an oncogene or tumor suppressor gene, depending on the specific type of cancer (8). It has been reported that elevated HOXB4 expression promotes ovarian cancer progression through encodes for the dehydrodolichyl diphosphate synthase subunit (DHDDS) and is associated with drug resistance (9,10). In colorectal cancer tissues, HOXB4 mRNA and protein levels are significantly elevated, and correlate with advanced pathological stage and poor survival outcomes (11). However, HOXB4 attenuates the tumorigenesis of cervical cancer cells by decreasing the activity of the Wnt/β-catenin signaling pathway (12), and inhibits breast cancer cell migration by targeting STARD13 (13). In hepatocellular carcinoma, HOXB4 functions as a tumor suppressor by negatively regulating the METTL7B/TKT axis. Downregulation of HOXB4 correlates with poor prognosis, while its overexpression suppresses proliferation, metastasis, and epithelial-mesenchymal transition (EMT) via downregulating mesenchymal markers (N-cadherin, Vimentin) and upregulating epithelial markers (E-cadherin) (14). In addition, some studies have focused on epigenetic alterations involving hypermethylation of the HOXB4 promoter in cancers, including LUADs (15-17), and it has recently been reported that identification of DNA methylation biomarkers, which includes HOXB4, provides the potential for early detection of lung cancers (18-20). Although these studies demonstrate that HOXB4 is involved in LUAD, its contribution to LUAD remains largely unknown.

In the present study, we therefore aimed to systematically determine the prognostic value, diagnostic value, and biological function of HOXB4 in LUAD through approaches including bioinformatics and experimental studies. We also sought to identify its molecular mechanisms of action. Our findings could provide novel insights into the treatment and diagnosis of LUAD. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-226/rc).

Methods

Cell culture and clinical specimens

The human LUAD cell line A549 was sourced from either the American Type Culture Collection (ATCC, Manassas, VA, USA) or collaborative partners. Cells were propagated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, under standard conditions (37 ℃, 5% CO₂). Primary tumor tissues, adjacent non-tumor tissues, and normal lung specimens were procured from the Department of Cardiothoracic Surgery at The First Affiliated Hospital of Chongqing Medical University (Chongqing, China; patient characteristics in Table 1). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The ethical approval for this study was obtained from the Clinical Trials Ethics Committee of The First Affiliated Hospital of Chongqing Medical University (Chongqing, China) (No. 2024-167-01). Written informed consent was taken from all the participants.

Analyses using online databases

Pan-cancer expression of HOXB4 was analyzed using the TIMER2 database (http://timer.cistrome.org/). The UALCAN (http://ualcan.path.uab.edu/) was used to analyze the expression and methylation levels of HOXB4 in normal and LUAD tissues as well as the relationship between HOXB4 DNA methylation and the clinical characteristics of LUAD patients. Then, we used MethMarkerDB (https://methmarkerdb.hzau.edu.cn/) to analyze the correlation between HOXB4 DNA methylation and patient survival in LUAD. Gene expression data were obtained from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/) and GSE37745 which was downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). Analysis was performed using R (version: 4.3.3) to obtain the most relevant genes of HOXB4 and uploaded to the Database for Annotation, Visualization, and Integrated Discovery (DAVID 2021). Finally, enrichment results were obtained from Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The top five results in ascending order of P value (P<0.05) were displayed in this study and mapped with the online web platform Sangerbox (http://www.sangerbox.com/tool).

RNA isolation and quantitative polymerase chain reaction (qPCR) assays

Total RNA was isolated from cells and tissues using TRIzol^® (Molecular Research Center, Cincinnati, OH, USA) per manufacturer’s protocol. Quantitative PCR employed SYBR® Green Master Mix (Thermo Fisher Scientific, Hong Kong, China) on an HT7500 system (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the endogenous control. Validated primers (e.g., HOXB4-F: 5'-CGTGAGCACGGTAAACCCC-3' and HOXB4-R: 5'-CGAGCGGATCTTGGTGTTG-3') were designed and validated for amplification efficiency. All primer sequences are listed in Table 2.

Construction of vector- and HOXB4-expressing stable cell lines

A HOXB4-expressing stable cell line was generated. First, a HOXB4-expressing plasmid was constructed by inserting HOXB4 full-length gene with a flag into pcDNA3.1(+) framework. The recombinant plasmid was sequenced. Next, pcDNA3.1 and HOXB4-containing plasmid (4 µg) were transfected into A549 cell line using Lipofectamine 2000 (Invitrogen, Carlsbad, USA). After transfection, cells were cultured in non-selective medium for 48 h before switching to selection medium containing 400 µg/mL G418 (Invitrogen/Gibco). After 14 days, individual colonies were isolated and cultured. Concurrently, DNase I-treated total RNA from transfected cells was used to confirm HOXB4 expression in stable cells via reverse transcription qPCR (RT-qPCR) and Western blotting.

Cell Counting Kit-8 (CCK-8) assay

Post-transfection with HOXB4 or control (pcDNA3.1) plasmids, A549 cells were plated at 2,000 cells/well in 96-well plates. Cell proliferation was assessed using the CCK-8 assay at 0, 24, 48, and 72 h. At each time point, 10 µL CCK-8 solution mixed with 100 µL DMEM complete medium was added per well. Plates were incubated for 2 h at 37 ℃ before measuring absorbance at 450 nm using a microplate reader. Three independent replicates were performed.

Cell migration and invasion assays

Cell migration and invasion were assessed via Transwell chambers (6.5 mm inserts, 8 µm pores). For invasion assays specifically, the membrane was coated with Matrigel (BD Biosciences, San Jose, CA, USA). This coating models the extracellular matrix barrier that invasive cells must degrade, distinguishing invasion from simple migration measured on uncoated membranes. Excess stain was washed off with PBS after 24 h fixation and staining, and cells in the upper chamber were wiped off pictured and tallied below microscope.

Flow cytometric analysis of cell cycle and apoptosis

To evaluate the cell cycle, cells were digested with trypsin and fixed with ice-cold 70% ethanol, treated with 5 mg/mL RNase A (Sigma, St. Louis, MO, USA), and stained with propidium iodide (PI). To analyze apoptosis, double staining was performed with annexin V-fluorescein isothiocyanate and PI. Data were analyzed using CellQuest™ Pro (BD Biosciences). All experiments were performed in triplicate.

Western blot

Cells underwent lysis using a protein extraction reagent (Thermo Scientific, Rockford, IL, USA) supplemented with the protease inhibitor phenylmethanesulfonyl fluoride (PMSF) and a phosphatase inhibitor cocktail (Sigma). Subsequently, proteins within the resultant lysates (50 µg total protein per sample) were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were then electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Following transfer, the membranes were subjected to incubation with specific primary antibodies sourced from Santa Cruz Biotechnology (Santa Cruz, CA, USA), targeting the following proteins: HOXB4 (sc-271083), RAP1; (sc-53434), RAP1GAP (sc-166586), E-cadherin (sc-8426), N-cadherin (sc-59987), Vimentin (sc-373717), and GAPDH (sc-32233). Finally, protein bands were detected and visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Statistical analysis

Statistical analyses and the production of graphs were performed with GraphPad Prism 9.1.0 (GraphPad Software, Inc., San Diego, CA, USA). All data are representative of three independent experiments and using Student t-test, or one-way analysis of variance with post hoc test. For all tests, data were considered significant at P<0.05.

Results

HOXB4 is a downregulated and hypermethylated gene in LUAD

Previously, we performed genome profiling using an 850K array and RNA sequencing in NSCLC tissue samples. Next, related heat and volcano maps were constructed (Figure 1A,1B). Hypermethylation of HOXB4 in NSCLC was also identified and verified.

Figure 1 HOXB4 expression and DNA methylation in lung adenocarcinoma tissues. (A) Cluster analysis of the Illumina Human Methylation 850K Microarray in 3 pairs of NSCLC tissues (heat map). (B) Cluster analysis of the Illumina Human Methylation 850K Microarray in 3 pairs of NSCLC tissues (volcanic map). (C) The expression differences of HOXB4 in cancer tissues and adjacent tissues of different cancer species in the TIMER2 database. (D) HOXB4 expression in Lung adenocarcinoma in the UALCAN database. (E) HOXB4 DNA methylation in lung adenocarcinoma in the UALCAN database. (F) HOXB4 DNA methylation was negatively correlated with HOXB4 transcript expression in the MethMarkerDB database. (G) The expression of HOXB4 in LUAD tissues and non-tumor adjacent surgical margin by RT-qPCR. *, P<0.05; **, P<0.01; ***, P<0.001. HOXB4, homeobox B4; LUAD, lung adenocarcinoma; NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription quantitative polymerase chain reaction; TCGA, The Cancer Genome Atlas.

To understand the function of HOXB4, we first examined this gene in the TIMER2 database to initially determine the presence of HOXB4 mRNA in human malignancies. We identified HOXB4 expression in 36 carcinomas, which showed a significant decrease in expression in LUAD, when compared with normal tissues (Figure 1C). Additionally, we found that HOXB4 expression was lower in LUAD tissues than in normal lung tissues found in the UALCAN database (Figure 1D). Moreover, the HOXB4 promoter CpG island was highly methylated, when compared with normal tissues (Figure 1E). Furthermore, analysis revealed a significant negative correlation between mRNA levels and methylation levels of HOXB4 (Pearson’s r: −0.505, P=8.7e−33; Figure 1F) in the MethMarkerDB database. We also determined the expression of HOXB4 mRNA using RT-PCR in 40 paired samples of human LUAD tissues and their surgical margins, which showed that HOXB4 expression was downregulated in primary LUAD tissues, when compared with their corresponding adjacent tissues (P=0.02; Figure 1G). Taken together, these results indicated that promoter cytosine-phosphate-guanine (CpG) island hypermethylation of HOXB4 was the underlying mechanism of its downregulation in LUADs, suggesting that HOXB4 could play a significant regulatory role in the development of LUAD.

HOXB4 methylation and its clinical relevance in LUADs

We then identified relationships between clinicopathological parameters and HOXB4 DNA methylation in LUAD patients using the online UALCAN database, which showed that the degree of HOXB4 methylation was positively correlated with tumor stage (Figure 2A). Moreover, the HOXB4 methylation level was significantly higher in Asians than in other races (Figure 2B). We also determined the distribution of differentially methylated regions around the HOXB4 gene in LUADs (Figure 2C), and further analyzed the association of HOXB4 DNA methylation with survival and prediction of tumors in LUAD patients using the MethMarkerDB database. The results confirmed that the high methylation levels of HOXB4 were associated with poor survival in LUADs (P=0.03; Figure 2D) and that its methylation level predicted the occurrence of LUADs [area under the curve (AUC): 0.974; 95% confidence interval: 0.94–1.00; Figure 2E].

Figure 2 Clinical significance of HOXB4 hypermethylation in LUAD. (A) The promoter methylation level of HOXB4 was related to the LUAD patient stage in UALCAN database. (B) The promoter methylation level of HOXB4 was related to LUAD patient race in UALCAN database. (C) The DMRs in the region 3 kb upstream and 3 kb downstream around the HOXB4 gene in lung adenocarcinoma. (D) AUC of ROC curves verified the diagnosis performance of HOXB4 DNA methylation in the MethMarkerDB database. (E) Association between HOXB4 DNA methylation and OS in the MethMarkerDB database. AUC, area under the curve; CI, confidence interval; DMR, differentially methylated region; HOXB4, homeobox B4; HR, hazard ratio; LUAD, lung adenocarcinoma; OS, overall survival; ROC, receiver operating characteristic; TCGA, The Cancer Genome Atlas.

Together, these results suggested that hypermethylation of HOXB4 was a risk factor for poor prognoses of LUAD patients, indicating that the presence of methylated HOXB4 could be used as a biomarker for LUAD prediction and prognosis.

HOXB4 overexpression inhibited LUAD cell proliferation, migration, and invasion

To test the possibility that HOXB4 may be a potential tumor suppressor gene in LUADs, we transfected a pcDNA3.1-Flag-HOXB4 expressing plasmid into the A549 LUAD cell line, resulting in high levels of expression. Restoration of HOXB4 expression after stable transfection was verified using RT-qPCR (Figure 3A). The tumor growth suppressive effects of HOXB4 protein on A549 (P<0.05) were confirmed using a CCK-8 assay (Figure 3B). As previously mentioned, metastasis is usually the main reason for unsatisfactory treatment outcomes and poor prognoses of LUAD patients. Thus, we evaluated the effect of HOXB4 protein on LUAD metastasis. Transwell assays were conducted to determine the effects of HOXB4 on cell migration and invasion, which showed that HOXB4 overexpression in A549 cells resulted in significant reductions in cell migration and invasion (Figure 3C,3D).

Figure 3 Overexpression of HOXB4 in A549 cell line inhibits cell proliferation and cell invasive migration. (A) RT-qPCR confirmed the overexpression of HOXB4 in A549 cell line (vector vs. HOXB4, P=0.049). (B) CCK-8 assay for cell proliferation on vector- and HOXB4-transfected cell lines. (C) Cell migration and invasion ability in vector control and HOXB4-expressing A549 cell lines by transwell assay (staining methods: 0.1% crystal violet; magnification: 10×). (D) Quantitative analysis of cell migration and invasion in vector and HOXB4-expressing A549 cell lines. *, P<0.05; ***, P<0.001. CCK-8, Cell Counting Kit-8; HOXB4, homeobox B4; OD, optical density; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

HOXB4 overexpression increased LUAD cell apoptosis and promoted cell cycle arrest

We then assessed whether HOXB4 affected cell apoptosis and cell cycle distribution of LUAD cells, using flow cytometry analysis. After transient transfection of a HOXB4 overexpression plasmid into A549 cells for 48 h, the cells were collected for detection of cell apoptosis and cell cycle distribution. The results showed that HOXB4 overexpression in A549 cells significantly increased cell apoptosis (Figure 4A,4B) and caused more cells to arrest in the G0/G1 phase (Figure 4C,4D). Taken together, the results showed that HOXB4 may be a tumor suppressor gene in LUAD, where it regulated proliferation, invasion, apoptosis, and the cell cycle.

Figure 4 The overexpression of HOXB4 induces apoptosis and cell cycle G0/G1 arrest in A549 cell lines. (A) HOXB4 induction of apoptosis detected by flow cytometric analysis with Annexin V-FITC and PI-staining in A549 cells. (B) Quantitative analysis of apoptosis. (C) Representative distribution of A549 cell lines both in vector and HOXB4 transfected cells. (D) The distribution and percentage of cells in the G0/G1, G2/M, and S phases of the cell cycle are indicated. **, P<0.01; ***, P<0.001. FITC, fluorescein isothiocyanate; HOXB4, homeobox B4; PI, propidium iodide.

Enrichment analysis of the HOXB4 gene in LUAD

To identify biological functions related to HOXB4, the genes most related to HOXB4 were screened using Pearson’s correlation analyses (|R| >0.5, P<0.05) in the TCGA and GEO databases, with the analyses based on the abovementioned gene sets. In the TCGA database, biological processes most related to HOXB4 included anterior/posterior pattern specification, embryonic skeletal system morphogenesis, and proximal/distal pattern formation (Figure 5A). Moreover, the cellular components most related to HOXB4 were chromatin (Figure 5B). The molecular functions involved RNA polymerase II transcription factor activity (Figure 5C). The HOXB4-related biological processes, cellular components, molecular functions in the GSE37745 database were similar to those in TCGA (Figure 5D-5F). Furthermore, analyses of both databases identified that the most related signaling pathway with HOXB4 is the Rap1 signaling pathway, including proteoglycans in cancer and axon guidance (Figure 5G,5H).

Figure 5 GO and KEGG pathway enrichment analysis of HOXB4-related genes in LUAD. (A-C) BP, CC, and MF are mostly related to HOXB4 in the TCGA database. (D-F) BP, CC, and MF are mostly related to HOXB4 in the GSE37745 database. (G) KEGG pathway analysis of HOXB4 in the TCGA database. (H) KEGG pathway analysis of HOXB4 in the GSE37745 database. BP, biological process; CC, cellular components; FDR, false discovery rate; GO, Gene Ontology; HOXB4, homeobox B4; KEGG, Kyoto Encyclopedia of Genes and Genomes; LUAD, lung adenocarcinoma; MF, molecular functions; TCGA, The Cancer Genome Atlas.

HOXB4 inhibited the EMT in LUAD cells by regulating the RAP1 signaling pathway

We selected A549 cells for the determination of downstream mechanisms, and found that the protein expression of the RAP1GAP and the E-cadherin epithelial marker was significantly elevated in the HOXB4 overexpression group, whereas protein expressions of the RAP1 and N-cadherin、vimentin mesenchymal markers were significantly reduced (Figure 6A,6B). In addition, RT-qPCR was performed to identify key genes of the RAP1 signaling pathway, which showed that overexpression of HOXB4 repressed the transcription of RAP1A/B and RAPGEF3/4 and increased RAP1GAP transcript levels in A549 cells (Figure 6C). Together, these findings suggested that HOXB4 regulated LUAD development by the EMT and through its involvement in the repression of the RAP1 signaling pathway (Figure 6D).

Figure 6 Overexpression of HOXB4 affects the RAP1 signaling pathway and EMT-related markers. (A) Western blot to detect the expression of key gene proteins in the RAP1 signaling pathway and EMT-related markers in HOXB4-expressing A549 cell lines. (B) Quantitative analysis of western blots. (C) RT-qPCR to detect RAP1 signaling pathway key genes expression in A549 cell after HOXB4 overexpression. (D) Schematic representation of the possible pathogenic mechanism of the HOXB4/RAP1 axis in LUAD progression. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant (P>0.05). The exact P values are presented in the Table S1. E-Cad, E-cadherin; EMT, epithelial-mesenchymal transition; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HOXB4, homeobox B4; LUAD, lung adenocarcinoma; N-Cad, N-cadherin; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Discussion

HOXB4 plays an important role in cancer proliferation, metastasis and angiogenesis (9,10,12,13,21-28). Our results showed that HOXB4 expression was downregulated and hypermethylated in LUAD tissues, while HOXB4 DNA methylation was associated with the prognosis and early diagnosis of LUAD. Overexpression of HOXB4 inhibited cell proliferation, migration, and invasion, and facilitated apoptosis in LUAD cells in vitro, while increasing cell cycle arrest. KEGG pathway analysis suggested that HOXB4-related genes were mainly clustered in the RAP1 pathway in LUAD patients. Together, our results suggested that HOXB4 inhibited malignant progression of LUAD cells by regulating the RAP1 signaling pathway to suppress the EMT in LUAD cells.

We also found that expression of HOXB4 was significantly reduced in LUAD tissues and cells. By altering the expression of HOXB4 in A549 cells, we found that high expression of HOXB4 significantly inhibited migration and invasion, as well as suppressed cell proliferation, and blocked the cell cycle transition from the G0/G1 to the S phases. Previous studies have reported that HOXB4 is frequently downregulated in cervical cancer cells, thereby promoting cell proliferation and tumorigenesis (12). In addition, Zhou et al. also reported that HOXB4 expression was downregulated in breast cancer cells, where it inhibited breast cancer cell migration in a STARD13-dependent manner (13). It has also been suggested that HOXB4 has anti-tumor effects in cancer, which is consistent with our findings. We therefore hypothesize that down-regulation of HOXB4 expression may act as an oncogenic gene effect in LUAD. We also showed that the methylation of HOXB4 DNA was significantly elevated in LUAD and negatively correlated with its expression level. Furthermore, DNA methylation of HOXB4 was negatively correlated with the prognosis of LUAD, and predicted the occurrence of LUAD (17). In recent years, accumulating evidence has demonstrated that DNA methylation markers play critical roles in the early diagnosis, therapeutic management, and prognostic evaluation of LUAD. Recent study has revealed that plasma-based detection of SHOX2 and RASSF1A methylation exhibits robust diagnostic performance for early-stage LUAD, achieving 61.11% sensitivity and an AUC of 0.806, thereby serving as a viable alternative to invasive bronchoalveolar lavage fluid (BALF) analysis (29). For prognostic evaluation, genome-wide methylation profiling via enzymatic methyl sequencing (EM-seq) delineates distinct epigenetic signatures in early LUAD. Prognostically significant markers such as ANLN and S100A16, coupled with a six-gene methylation-expression risk stratification model (log-rank P<0.0001), effectively categorize patients into high- and low-risk subgroups, guiding adjuvant therapy decisions (30). In the realm of therapeutic monitoring, tumor methylated fraction (TMeF) dynamics in circulating tumor DNA (ctDNA) correlate with pathological stage, histologic aggressiveness, and lymphovascular invasion, reflecting tumor-derived DNA shedding (31). These findings indicate that methylation markers play an important role in the diagnosis, treatment, and prognosis of LUAD. Notably, it has reported that seven differentially methylated genes, including HOXB4 in plasma, could be used for noninvasive differentiation between lung and non-lung cancers (19). In addition, Li et al. (20) developed a DNA methylation test using BALF samples containing the HOXB4 gene, for the diagnosis of malignant lung nodules, which is consistent with our findings, and suggested that the methylation of HOXB4 could be used to predict the progression of LUAD.

The EMT is important during tumor metastasis, because epithelial cells lose adhesion and gain invasive and metastatic capabilities (32-36). Cancer cells undergoing the EMT exhibit morphological changes and molecular alterations such as decreased expressions of epithelial markers, including E-cadherin, ZO-1, and occludin, and increased expressions of mesenchymal markers, including N-cadherin, vimentin, fibroblast-specific protein 1, and fibronectin (37). Previous study has reported that overexpression of HOXB4 promotes EMT-related metastasis in ovarian cancer (9). In addition, HOXB4 is an EMT-related gene, whose methylation has predictive value in lung cancer, and can be used as a marker for treatment options (17). Our western blot results showed that HOXB4 protein overexpression up-regulated expression of the E-cadherin epithelial marker, and down-regulated expressions of the N-cadherin and vimentin mesenchymal markers, which inhibited LUAD cell migration and invasion.

Although the role of HOXB4 in LUAD progression has been investigated, the specific mechanism by which HOXB4 regulates LUAD remains unclear. Further KEGG enrichment analyses revealed that HOXB4-related genes were mainly clustered in the RAP1 signaling pathway, suggesting that the effect of HOXB4 on LUAD may be mediated by the RAP1 signaling pathway. RAP1, a member of the ras-like family of small guanosine triphosphate (GTP)-binding proteins, which has two isoforms, Rap1A and Rap1B, binds GTP or guanosine diphosphate (GDP) (38,39). RAP1 activity is positively regulated by guanine nucleotide exchange factors (GEFs) and negatively regulated by GTPase-activating proteins (GAPs) (40). Studies have reported that RAP1 expression is significantly upregulated in lung cancer, while downregulation of RAP1 expression reduces migration and invasion in NSCLC cell lines (41). In addition, overexpression of SOX9 in LUAD significantly activates the RAP1 signaling pathway and promotes cell invasion and migration (42). Zhang et al. reported that silencing of CD147 inhibits proliferation, migration, and invasion of LUAD cells by blocking the Rap1 signaling pathway (43). Most importantly, Morsi et al. find that HOXB4 is a transcription factor that directly binds to two conserved sites within the 3’UTR of the Rap1 gene, suppressing its expression by inhibiting transcription. Overall, HOXB4 may exert inhibitory effects in LUAD via the RAP1 signaling pathway, which plays an important role in LUAD development (44). In the present study, after overexpression of HOXB4 in LUAD cells, the expression levels of genes related to the RAP1 signaling pathway changed with changes in HOXB4 expression. Importantly, transcriptions of RAP1A(B) and RAPGEF3(4) were negatively correlated with HOXB4 expression, while the transcription of RAP1GAP also significantly increased with increasing HOXB4 expression, at the same time, western blot results showed that overexpression of HOXB4 protein upregulated RAP1GAP expression and downregulated RAP1 expression. It was therefore possible that increased expression of HOXB4 may have inhibited the EMT in LUAD cells, by suppressing the RAP1 pathway, thus acting as a tumor suppressor.

The present study only investigated invasion and migration in one type of cell. Therefore, a more rigorous approach using multiple mutant cell systems and in vivo experiments should be conducted in future studies. Furthermore, in studies of the molecular mechanism of the tumor suppressor function of HOXB4, more studies will be needed to find more direct evidence of HOXB4 involvement in the RAP1 signaling pathway.

Conclusions

In conclusion, the present study provided evidence that HOXB4 had a tumor suppressor role in LUAD, and that hypermethylation of HOXB4 can predict LUAD and be associated with a poorer prognosis, which regulated the EMT in LUAD through the RAP1 signaling pathway. Based on these findings, we suggest that HOXB4 may be a target for the diagnosis and treatment of LUAD.

Acknowledgments

The authors are most grateful to all participants in this study.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-226/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-226/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-226/prf

Funding: This work was supported by Chongqing Nature Science Foundation (Nos. 2024NSCQ-MSX1843 and 2024NSCQ-MSX217).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-226/coif). All authors report the funding from Chongqing Nature Science Foundation (Nos. 2024NSCQ-MSX1843 and 2024NSCQ-MSX217). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The ethical approval for this study was obtained from the Clinical Trials Ethics Committee of The First Affiliated Hospital of Chongqing Medical University (Chongqing, China) (No. 2024-167-01). Written informed consent was taken from all the participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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