KLF5 facilitates lung adenocarcinoma metastasis via regulating the epithelial-mesenchymal transition pathway through RHPN2

KLF5 facilitates lung adenocarcinoma metastasis via regulating the epithelial-mesenchymal transition pathway through RHPN2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article KLF5 facilitates lung adenocarcinoma metastasis via regulating the epithelial-mesenchymal transition pathway through RHPN2 Tao Zhang, Ruo-qi Wang, Yu-bin Yang, Wen-jun Zhao, Qi-dong Zhang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6755394/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Oct, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Background Distant metastasis is the primary reason shortening the survival time for patients with advanced lung adenocarcinoma. Transcription factor Kruppel-like factor 5 (KLF5) has been confirmed to facilitate the progression of lung adenocarcinoma. However, the certain mechanism by which KLF5 involves in the tumor metastasis of lung adenocarcinoma remains largely unclear. Methods Gpt-ho-binding Protein 2 (RHPN2) was screened as the potential downstream target gene for KLF5 via bioinformatics analysis, which closely participates in regulation of the epithelial mesenchymal transformation (EMT) pathway in lung adenocarcinoma. Western blot and immunohistochemistry assays were performed to examine RHPN2 expression in lung adenocarcinoma. In vivo and in vitro experiments were conducted to explore the regulatory role of RHPN2 on cell growth and metastasis of lung adenocarcinoma. Chromatin immunoprecipitation sequencing (ChIP-seq) was used to analyze the direct binding activity between the KLF5 and RHPN2 promoter regions. The luciferase activity assay was carried out to verify the transcriptional activation effect of KLF5 to RHPH2. Results RHPN2 was found to be highly expressed in lung adenocarcinoma and patients with highly RHPN2 expression showed a poor prognosis in lung adenocarcinoma. In vivo and in vitro , experiments identified that RHPN2 promoted the cell growth and metastasis, and activated the EMT pathway in lung adenocarcinoma. Machanismly, KLF5 could directly bind to the promoter regions of RHPN2 and up-regulate the expression of the latter in lung adenocarcinoma through translational activation. In addition, the rescue experiments confirmed that RHPN2 facilitated the progression of lung adenocarcinoma at least in a KLF5-dependent form. Conclusion Our study offers insights into the potential mechanisms of metastasis in lung adenocarcinoma and suggests RHPN2 as a potential therapeutic target. lung adenocarcinoma RHPN2 KLF5 EMT metastasis transcription Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction It was estimated 1,060,600 new lung cancer cases, accounting for 22.0% of all malignancies, and around 733,300 new lung cancer deaths, accounting for 28.5% of all deaths from malignant tumors, occurred in China in 2022 [ 1 ] . Lung adenocarcinoma, characterized by insidious onset and early distant metastasis, is the most common sub-type for lung cancer. Distant metastasis is the main reason reducing the survival time of patients with advanced lung adenocarcinoma. Despite significant progresses for the treatment of lung adenocarcinoma, the 5-year survival rate to advanced patients is still less than 20% [ 2 ] . The mechanism of distant metastasis of lung adenocarcinoma cells is not entirely understood. It is well known that metastasis of tumor cells, including lung adenocarcinoma, is a complex multi-step process involving changes in multi-dimensional network systems, for instance aberrant changes of genome, transcriptome, proteomics and metabolites [ 3 ] . In our prior research, the transcription factor Kruppel-like factor 5 (KLF5) was reported to facilitate the malignant progression of lung adenocarcinoma through its direct interaction with ubiquitin-specific peptidase 38 [ 4 ] . In a recent single-cell analysis of patients with both metastatic and non-metastatic tumors, researchers delved into the intricate regulatory network of transcription factors that are active at various stages of metastasis. Through this comprehensive investigation, KLF5 is identified as a pivotal regulator deeply involved in cancer metastasis progress [ 5 ] .Through constructing regulatory networks for transcription factors on breast cancer through bulk, single-cell and single-nucleus multi-omic techniques as well as spatial transcriptomics and multiplex imaging, the importance of KLF5 in basal-like tumors and luminal progenitors are elaborately investigated [ 6 ] . In addition, KLF5 is reported to function as a crucial role in mediating glutamine metabolism, thereby exerting a significant influence on tumor cell growth in non-small cell lung cancer [ 7 ] . Furthermore, KLF5 has been identified as a critical regulator involved in chemokine production and neutrophil recruitment in lung squamous cell carcinoma, thereby significantly influencing the tumor immune microenvironment [ 8 ] . Taken all into account, the impact of KLF5 on lung adenocarcinoma is both comprehensive and highly significant. However, the downstream molecular mechanism of KLF5 in lung adenocarcinoma remains largely unclear. In the current study, we identified GTP-HO-binding Protein 2 (RHPN2) as the downstream target gene of KLF5 by bioinformatics methods. Combined bioinformation analysis and tissue sample detecting identification RHPN2 is found highly expressed in lung adenocarcinoma and is a critical factor contributing to the poor prognosis of patients. In vitro and in vivo experiments revealed that RHPN2 is able to promote the growth and metastasis of lung adenocarcinoma cells. More importantly, we demonstrated that RHPN2 promotes the process of epithelial mesenchymal transformation (EMT) in lung adenocarcinoma cells in a KLF5-dependent form. Materials and methods Chromatin-immunoprecipitation (ChIP) -seq data analysis The KLF5 ChIP-seq data used in this study are available in the GENE EXPRESSION OMNIBUS (GEO) database under accession number GSE164852 [9] . Quality control of the raw FASTQ data was performed using FastQC (v0.11.9) [10] to assess data quality. Trimmomatic (v0.39) [11] was used to trim low-quality bases and adapter sequences, and reads shorter than 36 bp were filtered out. The clean reads were aligned to the hg19 reference genome using Bowtie2 (v2.4.4) [12] . The resulting SAM files were converted to BAM format using SAMtools (v1.12) [13] , and duplicate reads were removed using Picard tools (v2.25.0) [14] to reduce PCR amplification bias. Peak calling was performed using MACS2 (v2.2.7.1) [15] with the parameters -B, --qvalue 0.01, and --gsize hs to identify genomic binding sites. The output BED files contained peak locations and significance scores. Peaks were annotated using ChIPseeker (v1.30.3) [16] to analyze their distribution across genomic features (e.g., promoter regions, exons) and to associate them with the nearest genes using the TxDb.Hsapiens.UCSC.hg19.knownGene database. Peak distribution statistics were generated, and the data were visualized using the Integrative Genomics Viewer (IGV, v2.11.1) [17] to display binding peaks in the context of genomic annotations. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis GO and KEGG pathway enrichment analyses were performed using clusterProfiler (v4.2.2) [18] . Target genes were extracted based on the nearest genes to the peaks identified by ChIPseeker. For GO analysis, enrichment was performed for Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) categories, with a significance threshold of P <0.05 and Benjamini-Hochberg correction for multiple testing. For KEGG analysis, target genes were mapped to the KEGG pathway database, and significantly enriched pathways ( P <0.05) were identified. The Cancer Genome Atlas Program (TCGA) The data on lung adenocarcinoma in the TCGA database (https://portal.gdc.cancer.gov) were used to analyze the expression of RHPN2 and its relationship with the prognosis of patients. Tissue samples and cell lines Twenty tumor tissues and their corresponding acjacent normal tissues were collected from patients with lung adenocarcinoma to detect of RHPN2 expression. These patients were diagnosed with lung adenocarcinoma through pathology and received surgical treatment at The First Hospital of Lanzhou University from March to June 2020. They did not receive radiotherapy, chemotherapy or immunotherapy before the operation. All tissues were immediately frozen in liquid nitrogen after resection and then stored at -80°C. This study was approved by the Ethics Committee of The First Hospital of Lanzhou University in China (LDYYLL2022-448). Human normal lung epithelial cell line (BEAS-2B) and lung adenocarcinoma cell line (A549, H1299, H1975 and HCC827) were derived from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, NY, USA), which contained 1% penicillin/streptomycin (Invitrogen, CA, USA) and 10% fetal bovine serum (FBS; Gibco) added 5% CO 2 at 37°C, humidified incubator (Thermo Scientific, CA, USA). Hematoxylin and Eosin (H&E) staining and Immunohistochemical (IHC) staining The tissues were fixed with a 10% formaldehyde solution and then dehydrated with gradient ethanol. Briefly, the tissues were embedded with paraffin wax and then made into 4 μm continuous sections. First, immerse the slices in the hematoxylin staining solution for 3 to 5 minutes. After removing the excess hematoxylin with 1% hydrochloric acid alcohol, immerse the slices in eosin staining solution for 1-2 minutes. Dehydration is carried out again using gradient ethanol. Finally, use neutral gum to seal the slices. IHC staining was performed on the prepared tissue sections. The sections were soaked in 3% H 2 O 2 for 5 minutes at room temperature to remove endogenous peroxidase activity, then incubated in sodium citrate buffer at 95°C for 15 minutes to repair the antigen. The sections were blocked by 5% normal goat serum for 10 minutes to seal the non-specific binding site. Next, the indicated primary antibody was added and incubated at 4℃ overnight. Finally, the secondary antibody labeled with horseradish peroxidase was incubated for 1 hour. The IHC staining results were evaluated by combining the staining intensity and the proportion of tumor cells. The final immune response score was calculated as the staining intensity score × the percentage of positive cells. The immune response score of 0-4 indicates low expression, and 5-9 indicates high expression. The antibodies used in this study are listed in Table S1 . Lentivirus transduction Small hairpin RNA (shRNA) targeting KLF5 and RHPN2, and containing amplified human RHPN2 or KLF5 sequence vectors were synthesized by HANBIO Co., Ltd. (Shanghai, China) and packaged as lentivirus particles. Then, lentivirus was transfected into lung adenocarcinoma cells, and puromycin was used to screen the stably transfected cell lines. The shRNA sequences are listed in Table S2 . Western blot Proteins in cells or tissues were extracted using radioimmunoprecipitation assay buffer (RIPA, Beyotime, Shanghai, China), and quantified using the biuretic acid assay kit (BCA, Beyotime) in accordance with the manufacturer's instructions. The protein was transferred onto the polyvinylidene fluoride (PVDF) membrane (Millipore, Boston, MA, USA) and incubated overnight with the primary antibody targeting the target protein at 4°C. The next day, the membrane was incubated with the indicated secondary antibody at room temperature for 1 hour. The immunoreactivity was observed using the automatic chemiluminescence image processing system (Bio-Rad, San Jose, CA, USA). The antibodies used in this study are listed in Table S1 . Cell viability assay Cell counting kit 8 (CCK8, Yelasen, Shanghai, China) and colony formation assay was used to detect the cell viability of lung adenocarcinoma cells, which was performed as described previously [4] . Transwell assay and wound healing assay The transwell assay and wound healing assay were used to evaluate the cell migration ability of lung adenocarcinoma cells, which was performed as described previously [19] . RT-qPCR RT-qPCR was performed as described previously [19] . The primer sequences are listed in Table S3 . Immunofluorescence assay (IF) Immunofluorescence assay was performed as described previously [11] . The antibodies used in this study are listed in Table S1 . JASPAR The JASPAR database (https://jaspar.genereg.net/) [20] was used to identify the binding site of transcription factor KLF5 to the RHPN2 promoter. ChIP-qPCR According to the manufacturer's instructions, ChIP assay was conducted using the SimpleChIP® Enzymatic Chromatin IP Kit (CST, Massachusetts, MA, USA). In detail, A549 cells were fixed with 1% paraformaldehyde for 15 minutes, then 0.125M glycine was added and quenched for 10 minutes. A lysis buffer containing protease inhibitors was added to the cells, and chromatin fragments were generated using an ultrasonic fragmentation device. The cleaved chromatin was incubated overnight with KLF5 antibody (Proteintech, Wuhan, China) and Protein G magnetic beads at 4°C. Rabbit IgG (CST, Massachusetts, MA, USA) was served as a negative control. After washing and elution, the purified DNA was used for qPCR analysis. The primers sequences for ChIP-qPCR are listed in Table S4 . Luciferase activity assay Luciferase activity assay was performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, wild-type (WT) and mutant (Mut1) RHPN2 promoter sequences were amplified and cloned into the pGL4.10-Basic vector (Promega), generating constructs named pGL4.10-RHPN2-promoter and pGL4.10-RHPN2-Mut1-promoter, respectively. The A549 cell line was co-transfected with these promoter constructs along with either pcDNA3.1-KLF5 expression plasmid or control pcDNA3.1 vector using Lipofectamine 3000 (Invitrogen, USA). Cells were cultured for 48 hours post-transfection. Luciferase activity was measured using a luminometer (GloMax, Promega), and results were normalized to Renilla luciferase activity as an internal control. Animal model week-old male BALB/c nude mice (Ziyuan Laboratory of Animal Science and Technology, Hangzhou, China) were used to construct animal models. Nude mice were randomly divided into the following 4 groups (n=5 each): sh-NC, sh-RHPN2#3, sh-KLF5, and sh-KLF5+oeRHPN2. The subcutaneous tumorigenesis model of nude mice was used to simulate the growth of tumors in vivo . 4×10 6 A549 cells were inoculated subcutaneously into each mouse, and the volume of the tumor was measured weekly using a vernier caliper. The calculation formula for tumor volume is as follows: volume = (length × width 2 )/2. The mice were euthanized after four weeks and the tumor tissue was collected and weighed. Then the tumor tissues were immobilized with formalin for subsequent analysis. An animal model of tumor metastasis i n vivo was simulated by injecting 3×10 6 A549-luc cells through the tail vein of nude mice. The bioluminescence images were acquired by the IVIS spectrum imaging system (PerkinElmer, Massachusetts, USA). Five weeks later, the mice were euthanized and their lung tissues were dissected. Then, the number of metastatic nodules on the surface of the lung tissues was calculated. Animal experiments were reviewed and approved by the Ethics Committee of The First Hospital of Lanzhou University. All animals were cared for in accordance with the Guide for the “Care and Use of Laboratory Animals”. Statistical Analysis Continuous variables were expressed as mean ± standard deviation. Differences between two groups were assessed using the independent samples t -test. For comparisons among three or more groups, one-way analysis of variance (ANOVA) was employed, followed by the least significant difference (LSD) test for pairwise comparisons. The correlation between continuous variables was assessed using Pearson's correlation analysis, with R representing the correlation coefficient. All statistical analyses and graphical representations were performed using GraphPad Prism version 8.0 and R software (version 4.4.1). P value < 0.05 was considered statistically significant. Results KLF5 ChIP-seq analysis in lung adenocarcinoma cells reveals key regulatory roles The genomic binding profile of KLF5, a transcription factor implicated in lung adenocarcinoma, was investigated through ChIP-seq analysis. The distribution of KLF5 binding peaks shows a higher-level enrichment at promoter regions and distal intergenic regions, consistent with its role as a transcription factor (Fig. 1 A). This observation is further supported by the concentration of binding peaks within ± 0.5 kb of transcription start sites (TSS), indicating that KLF5 primarily regulates gene expression by binding near promoter regions (Fig. 1 B). Functional annotation of KLF5 binding peak-annotated genes through GO and KEGG pathway analysis provides insights into the biological processes and pathways regulated by KLF5 in lung adenocarcinoma cells. The BP analysis highlights KLF5's involvement in critical cellular functions such as cell migration (ameboidal-type, endothelial, and epithelial), epithelial cell proliferation, and tissue morphogenesis, processes closely linked to cancer progression and metastasis. The CC analysis reveals associations with key cellular structures, including adherens junctions, focal adhesions, and membrane microdomains, which are essential for cell-cell communication and signaling. The MF analysis further supports KLF5's role in transcriptional regulation, with enriched terms such as DNA-binding transcription activator activity and RNA polymerase II-specific binding. Additionally, KEGG pathway analysis identifies significant enrichment in pathways related to cancer progression, including the Hippo signaling pathway, Rap1 signaling pathway, and small cell lung cancer, suggesting that KLF5 may play a essential role on modulating these oncogenic pathways (Fig. 1 C). Among those genes with higher KLF5 binding signals, five (ADAP1, CTNNA1, DUSP26, MYH14, and RHPN2) are highlighted as potential direct transcriptional targets of KLF5 (Fig. 1 D). Notably, RHPN2 shows a significant positive correlation with KLF5 expression, with a correlation coefficient of 0.38 and a P -value < 0.001, suggesting a potential regulatory relationship between KLF5 and RHPN2 in lung adenocarcinoma cells (Fig. 1 E). Collectively, these findings emphasize KLF5's central role on regulating genes and pathways associated with lung adenocarcinoma progression, particularly in processes related to cell migration, proliferation, and other oncogenic signaling pathways. The identification of KLF5 binding peak-annotated genes and their functional annotations provide a foundation for further exploration of KLF5's mechanistic contributions to lung adenocarcinoma biology. RHPN2 is highly expressed in lung adenocarcinoma As previously mentioned, RHPN2 is a potential target gene for the transcription factor KLF5, while its role on the progression of lung adenocarcinoma is unclear. Based on the data analysis for lung adenocarcinoma in the TCGA database, RHPN2 expression was found up-regulated in lung adenocarcinoma samples compared to normal samples ( Fig. 2 A ) . Moreover, High RHPN2 expression is associated with shorter survival in patients with lung adenocarcinoma (Fig. 2 B). Univariate and multivariate analysis suggested that high expression of RHPN2 was an independent risk factor for poor prognosis of lung adenocarcinoma (Fig. 2 C-D). Next, we detected the expression of RHPN2 in lung adenocarcinoma cell lines and tissues by western blot and IHC, respectively. Compared with human bronchial epithelial BEAS-2B cell line, RHPN2 expression was significantly up-regulated in lung adenocarcinoma cell lines (Fig. 2 E). Among them, the A549, H1299, and H1975 cell lines were used in the subsequent experiments. In addition, the expression level of RHPN2 in lung adenocarcinoma tissues was significantly higher than that in adjacent tissues (Fig. 2 F). At the same time, RHPN2 protein was mainly located in the cytoplasm of lung adenocarcinoma cells through IHC detection (Fig. 2 G). In summary, RHPN2 is highly expressed in lung adenocarcinoma and is an independent risk factor for predicting poor prognosis. RHPN2 promotes the growth of lung adenocarcinoma To explore the effect of RHPN2 on the progression of lung adenocarcinoma, we first constructed a RHPN2-deficient lung adenocarcinoma cell lines. As shown in Fig. 3 A, the inhibition rates in RHPN2 lung adenocarcinoma cell lines A549 and H1299 were verified by western blot. Based on the western blot results, sh-RHPN2#2 and #3 showed better inhibitory effects and were therefore used in subsequent functional studies. Next, we focused on investigating the effect of RHPN2 on the cell proliferation of lung adenocarcinoma cells through the CCK-8 and colony formation assays. The results of CCK-8 assay showed that knockdown of RHPN2 could lead to a significant decrease in the cell viability of A549 and H1299 cells (Fig. 3 B). The colony formation experiments also showed similar trends and results (Fig. 3 C). Meanwhile, we constructed a subcutaneous tumor model in nude mice using the A549 cell line (sh-RHPN2#3) to verify the effect of RHPN2 on the growth of lung adenocarcinoma in vivo . Compared with the control group, the volume and weight of subcutaneous xenografts in nude mice in the sh-RHPN2#3 group were significantly reduced (Fig. 3 D-E). Histological analysis by H&E staining confirmed that the xenografts were indeed tumor tissues (Fig. 3 F). Finally, we detected the expression changes of the cell proliferation marker Ki-67 protein in the xenograft tissues through IHC, and found that knockdown of RHPN2 could inhibit its expression (Fig. 3 G). Overall, RHPN2 may promote the cell proliferation in vitro and tumor growth in vivo for lung adenocarcinoma. RHPN2 facilitates tumor metastasis of lung adenocarcinoma Given the clinical association of RHPN2 with tumor metastasis of lung adenocarcinoma, we then focused on studying its role in regulating the migration and invasion of lung adenocarcinoma cells. Transwell and wound healing assays showed that knockdown of RHPN2 significantly inhibited the cell migration and invasion of A549 and H1299 cell lines, highlighting its critical role in lung adenocarcinoma metastasis (Fig. 4 A-B). Then, we analyzed the potential molecular biological mechanism of RHPN2 in the process of lung adenocarcinoma through Gene Set Enrichment Analysis (GSEA). The GSEA results indicated that RHPN2 was closely associated with multiple tumor-related pathways, including poor survival of lung cancer and EMT (Fig. 4 C). Among them, EMT is an important precursor step for distant metastasis of cancer cells. Therefore, we analyzed the effect of RHPN2 on the expression of EMT-related proteins in lung adenocarcinoma cell lines by western blot. The results showed that knockdown of RHPN2 could significantly inhibit the expressions of Snail, N-cadherin, and Vimentin, but up-regulated the expression level of E-cadherin (Fig. 4 D). Those results increase the favorable evidence that RHPN2 could promote the cell migration of lung adenocarcinoma cells. Finally, we constructed an animal model of in vivo metastasis of tumor cells by injecting lung adenocarcinoma cells into the tail vein of nude mice, and the successful modeling was proved by using fluorescence imaging (Fig. 4 F). Five weeks after injecting A549-luc cells into the tail vein of mice, we dissected their lungs and calculated the number of nodules on the lung surface, and confirmed that they were metastatic tissues through H&E staining. The results showed that knockdown of RHPN2 could significantly repress the metastasis of lung adenocarcinoma in nude mice (Fig. 4 G-H). The above-mentioned studies have proved that RHPN2 may facilitate the distant metastasis of lung adenocarcinoma. KLF5 transcriptionally upregulates RHPN2 expression in lung adenocarcinoma In previous studies, we have confirmed that RHPN2 is upregulated in lung adenocarcinoma and promotes the malignant phenotype of tumor cells. Next, we focused on investigating whether the high expression of RHPN2 was regulated by the transcription factor KLF5. RT-qPCR and western blot confirmed that knockdown of KLF5 could inhibit the expression level of RHPN2 in A549 cells, respectively, while overexpression of KLF5 further promoted the expression level of RHPN2 in H1975 cells (Fig. 5 A-B). Those results indicate that KLF5 has a positive regulatory effect on RHPN2 in lung adenocarcinoma. Then, we detected the localization of KLF5 and RHPN2 proteins in lung adenocarcinoma cells through IF staining. As shown in Fig. 5 C, the KLF5 protein was mainly located in the cell nucleus, and the RHPN2 protein was mainly located in the cytoplasm of lung adenocarcinoma cells. To evaluate whether the KLF5 protein could directly bind to the promoter regions of RHPN2 gene , we first used the JASPAR database to determine three sites where the RHPN2 ’s promoter region may bind to KLF5 (Fig. 5 D-E). We precisely detected the binding sites on those RHPN2 promoters by ChIP-qPCR. We detected the direct action of KLF5 protein on the binding sites of these RHPN2 promote regions in A549 cells by ChIP-qPCR. Results showed that the second site (-686 to -697) in the promoter regions of RHPN2 was an important binding site for the KLF5 protein (Fig. 5 F). Subsequently, in order to further verify the transcriptional activation effect of the transcription factor KLF5 on RHPN2, we conducted the dual-luciferase reporter gene assay using A549 cells. Based on the ChIP-qPCR results, we mutated the first site where the KLF5 protein mainly binds in the RHPN2 promoter region, and then detected the activity of luciferase. The luciferase activity of the wild-type RHPN2 promoter was significantly higher than that of the mutant RHPN2 promoters (Fig. 5 G). These results indicate that there is a positive regulatory relationship between the transcription factor KLF5 and its downstream target gene RHPN2. In conclusion, KLF5 promotes the expression of RHPN2 in lung adenocarcinoma through transcriptional upregulation. RHPN2 promotes the progression of lung adenocarcinoma in a KLF5-dependent form To further clarify that RHPN2 promotes the process of lung adenocarcinoma in a KLF5-dependent form, we conducted rescue experiments by overexpress RHPN2 in sh-KLF5 A549 cells and then examined the changes in cell proliferation and migration of lung adenocarcinoma. CCK8 and colony formation assays demonstrated that KLF5 knockdown inhibited the cell proliferation of A549 cells, meanwhile, overexpression of RHPN2 reversed part of this trend (Fig. 6 A-B). These results indicated that RHPN2 overexpression reversed the inhibitory role of KLF5 knockdown on tumor cell growth of lung adenocarcinoma (Fig. 6 C-D). Similarly, we performed IHC staining of Ki-67 on the tumor tissues. Results showed that knockdown of KLF5 inhibited the expression of Ki-67, while overexpression of RHPN2 reversed this trend (Fig. 6 E-F). Additionally, KLF5 knockdown inhibited the cell migration and invasion capacity of A549 cells, and the sh-KLF5-induced reduction of trend was partially rescued by co-transfection with RHPN2 in transwell and wound healing assays, (Fig. 7 A-B). Western blot revealed that the sh-KLF5-induced decrease in Snail, N-cadherin, and Vimentin partially reversed by the overexpression of RHPN2, and the sh-KLF5-induced increase in E-cadherin expression was also partially reversed by RHPN2 overexpression (Fig. 7 C). In the nude mouse model of lung adenocarcinoma metastasis in vivo , we observed that knockdown of KLF5 significantly reduced the number of lung metastatic nodules, while RHPN2 was found to reverse the inhibitory effect of sh-KLF5 on metastatic nodules (Fig. 7 D-F). Collectively, in vivo and in vitro studies have shown that RHPN2 promotes the growth and metastasis of lung adenocarcinoma in a form dependent on the transcription factor KLF5. KLF5 expression is positively correlated with RHPN2 in lung adenocarcinoma To further clarify the correlation between KLF5 and RHPN2 in lung adenocarcinoma, we detected the expression of both KLF5 and RHPN2 in 20 cases of lung adenocarcinoma tissues through IHC (Fig. 8 A). According to the scoring criteria of IHC, we statistically found that the expression level of KLF5 was significantly positively correlated with RHPN2 in lung adenocarcinoma (Fig. 8 B). Those results strengthen the evidence that RHPN2 is a key target of the transcription factor KLF5. Discussion Lung adenocarcinoma has an insidious onset and is characterized by distant metastasis at an early stage. Distant metastasis is an important factor contributing to a poor prognosis in patients with lung adenocarcinoma. The molecular mechanism of distant metastasis of lung adenocarcinoma remains unclear, and in-depth exploration of it will help improve the poor prognosis of patients. The EMT program, as a set of multiple and dynamic transition states between epithelial and mesenchymal phenotypes, has been clarified as an important precursor step for distant metastasis of cancer cells [ 21 ] . Multiple studies have confirmed the key role of the EMT pathway in the progression of lung adenocarcinoma. It is reported that CHIP-mediated CIB1 ubiquitination regulated EMT and tumor metastasis in lung adenocarcinoma [ 22 ] . In addition, cigarette smoke was identified to induce EMT, stemness, and metastasis in lung adenocarcinoma cells via upregulated RUNX-2/galectin-3 pathway [ 23 ] . In the present study, we first confirmed that RHPN2 was upregulated in lung adenocarcinoma and was closely associated with the activation of the EMT pathway. RHPN2 is a Rho GTPase-binding protein, which was initially discovered to be involved in the recombination of the cytoskeleton and the alteration of cell morphology [ 24 ] . Recent studies have shown that RHPN2 plays an important role on oncogenic signaling in various tumors, including promoting cell proliferation, migration and invasion of tumor cells [ 25 – 27 ] . Up to date, little is known for RHPN2 in lung adenocarcinoma. He et al. discovered that RHPN2 is necessary for cell proliferation and invasion of lung cancer cells. Interestingly, overexpression of RHPN2 endows lung cancer cells with resistance to glutamine depletion [ 28 ] . In the current study, we systematically analyzed the role of RHPN2 in lung adenocarcinoma through multiple methods. Based on bioinformatics analysis and tissue sample confirming, we found that RHPN2 was upregulated in lung adenocarcinoma and was closely associated with the poor prognosis of patients. Moreover, in vitro and in vivo experiments confirmed that RHPN2 could promote the growth and metastasis of lung adenocarcinoma. More importantly, our research clarified that RHPN2 promotes the metastasis of lung adenocarcinoma cells by regulating the EMT pathway. The above results highlight the importance of RHPN2 in the process of lung adenocarcinoma. However, the specific mechanism of RHPN2 in lung adenocarcinoma remains unclear. Herein, we identified that RHPN2 interacts with KLF5 to exert its oncogenic functions lung adenocarcinoma. Multiple studies have shown that KLF5 plays an important role in the progression of lung adenocarcinoma [ 29 ][ 30 ] . Our previous research found that the N-terminal of USP38 (residues 1-400aa) interacts with the residues 1-200 aa of KLF5, thereby stabilizing the KLF5 protein through deubiquitination [ 4 ] . Our analysis revealed that KLF5, as a transcription factor, could bind to different gene promoter regions through bioinformatics. Among them, RHPN2 is the target gene of KLF5 and is closely related to it in lung adenocarcinoma. Mechanically, we clarified that KLF5 affects the expression of RHPN2 in lung adenocarcinoma through transcriptional activation. Further rescue experiment confirmed that RHPN2 promotes the progression of lung adenocarcinoma in a KLF5-dependent manner, especially in terms of the regulation of the EMT pathway. These findings suggest that targeting the KLF5/RHPN2/EMT axis may be a promising therapeutic strategy for lung adenocarcinoma. Future research could explore small molecule inhibitors targeting the KLF5/RHPN2 axis to test their indirect regulation of the EMT pathway. This study also has certain limitations. For example, the exploration of the regulatory mechanisms of KLF5 and RHPN2 is relatively single, and it is debatable whether there are complexes or super enhancers affecting their interaction. In addition, the cell or animal models we selected are relatively simple. Future research could verify the effectiveness of these strategies under the condition of simulating the tumor microenvironment by using organoid models (PDO) and patient-derived xenotransplantation (PDX) models, thereby accelerating their clinical transformation process. This study revealed the regulatory role of RHPN2 in the growth and metastasis of lung adenocarcinoma, emphasizing that it regulates the EMT pathway in a KLF5-dependent manner (Fig. 8 C). This novel finding not only enhances our understanding of EMT signaling, but also provides new insights into the potential of targeting this pathway in the treatment of lung adenocarcinoma. Abbreviations KLF5, transcription factor Kruppel-like factor 5; RHPN2, GTP-HO-binding Protein 2; EMT, epithelial mesenchymal transformation; GEO, GENE EXPRESSION OMNIBUS; ChIP, Chromatin-immunoprecipitation; IGV, Integrative Genomics Viewer; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BP, Biological Process; MF, Molecular Function; CC, Cellular Component; TCGA, The Cancer Genome Atlas Program; RPMI, Roswell Park Memorial Institute; FBS, fetal bovine serum; H&E, Hematoxylin and Eosin; IHC, Immunohistochemical; shRNA, small hairpin RNA; RIPA, radioimmunoprecipitation assay buffer; BCA, biuretic acid assay; PVDF, polyvinylidene fluoride; CCK8, cell counting kit 8; IF, immunofluorescence assay; WT, wild-type; Mut, mutant; ANOVA, one-way analysis of variance; LSD, least significant difference; TSS, transcription start sites; GSEA, Gene Set Enrichment Analysis; PDO, organoid models; PDX, patient-derived xenotransplantation. Declarations Acknowledgements We thank Professor Ran-ran sun for his guidance and assistance in animal experiments. ChIP-qPCR service was provided by Shanghai Cutseq Biomedical Technology Co., Ltd. We also acknowledge TCGA, GEO and JASPAR database for providing their platforms and contributors for uploading their meaningful datasets. Author contributions TZ, RQW and YBY drafted the article and interpreted data. TZ, RQW, YBY, WJZ, ZQD and XLM performed the experiments and obtained the data. YBY and QS conducted bioinformatic analysis. JJG and QDZ collected clinical samples. XYS assisted in revising the format of the manuscript. YBL, FS, and XMH designed the study and revised the article critically for important intellectual content. All authors read and approved the final manuscript. Funding This work was funded by the (1) Youth Science and Technology Talent Innovation Project of Lanzhou City (2023-QN-14), (2) Medical Innovation and Development Project of Lanzhou University (lzuyxcx-2022-183), (3) Science and Technology Plan Project in Chengguan District of Lanzhou City (2021RCCX0008), (4) Key Research and Development Plan from Gansu Provincial Department of Science and Technology (22YF7FA086), (5) Unite Pesearch Foundation of Gansu Province (23JRRA1497), (6) Natural Science Foundation of Gansu Province (23JRRA0928 and 23JRRA1607), (7) Key Talent Project of Gansu Province (2025RCXM036). Data availability No datasets were generated or analysed during the current study. Competing interests The authors declare no competing interests. References Han B, Zheng R, Zeng H, Wang S, Sun K, Chen R, et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent. 2024;4(1):47–53. Reck M, Heigener DF, Mok T, Soria JC, Rabe KF. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6755394","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":469463325,"identity":"d160ac18-d6ab-407c-a18e-2d3bad52aade","order_by":0,"name":"Tao Zhang","email":"","orcid":"","institution":"Lanzhou University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Zhang","suffix":""},{"id":469463326,"identity":"afdf6efd-a9d6-48ec-be51-61bad463d89f","order_by":1,"name":"Ruo-qi Wang","email":"","orcid":"","institution":"Gansu University of Chinese 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05:02:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6755394/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6755394/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-025-07150-6","type":"published","date":"2025-10-10T15:57:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84531874,"identity":"d796c762-3ef1-423c-adcd-f5b487778b22","added_by":"auto","created_at":"2025-06-13 06:19:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4396947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic Binding Profile and Functional Annotation of KLF5 in Lung Cancer Cells. \u003c/strong\u003e(A) Distribution of KLF5 binding peaks across genomic regions, including promoter regions, distal intergenic regions, and other annotated genomic features. Peaks were annotated using ChIP seeker, and the distribution is presented as a percentage of total peaks. (B) Binding peaks are predominantly concentrated within ± 0.5 kb of transcription start sites (TSS), as determined by peak calling using MACS2 (q-value \u0026lt; 0.01). The frequency of peaks relative to TSS is shown. (C) Functional annotation of KLF5-bound genes through GO and KEGG pathway analysis. GO terms (BP, CC, MF) and KEGG pathways were analyzed using clusterProfiler, with significance thresholds set at \u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.05 after Benjamini-Hochberg correction. The top enriched terms and pathways are displayed. (D) Five genes (ADAP1, CTNNA1, DUSP26, MYH14, and RHPN2) with higher KLF5 binding signals (top 5% of peaks based on peak score) are shown. (E) Correlation analysis between KLF5 and RHPN2 expression, calculated using Pearson correlation (R=0.38, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Correlations were analyzed using the Pearson method and the log2-transformed TPM values were used for the analysis.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755394/v1/3d8a3016f8badd586dd592f5.jpg"},{"id":84531881,"identity":"5df39218-290b-484d-be76-79f8e48ca1f3","added_by":"auto","created_at":"2025-06-13 06:19:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2600857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRHPN2 is upregulated in lung adenocarcinoma and \u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eis\u003c/strong\u003e\u003c/del\u003e\u003cstrong\u003e closely related to the poor prognosis of patients.\u003c/strong\u003e (A) The expression of RHPN2 in normal samples and lung adenocarcinoma samples was analyzed based on the TCGA dataset. (B) The relationship between the expression of RHPN2 and the prognosis of patients with lung adenocarcinoma was analyzed based on the TCGA dataset. (C) Forest plot for univariate regression analysis of prognosis in patients with lung adenocarcinoma based on TCGA data. (D) Forest plot for multivariate regression analysis of prognosis in patients with lung adenocarcinoma based on TCGA data. (E) Western blot was used to detect the expression level of RHPN2 protein in human bronchial epithelial BEAS-2B cell line and lung adenocarcinoma cell lines. (F) Western blot was used to analyze the expression level of RHPN2 protein in five pairs of adjacent tissues and lung adenocarcinoma tissues. (G) The expression of RHPN2 protein in adjacent tissues and lung adenocarcinoma tissues was detected by IHC. * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755394/v1/62080af1521348d7781a086b.jpg"},{"id":84531875,"identity":"6e929568-3727-4179-acbc-d3ba2b347c4e","added_by":"auto","created_at":"2025-06-13 06:19:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3055155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of RHPN2 on the growth of lung adenocarcinoma was analyzed through\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e experiments.\u003c/strong\u003e (A) Western blot was used to identify the inhibitory rate of RHPN2 in lung adenocarcinoma cell lines. (B) The effect of RHPN2 on cell viability of lung adenocarcinoma cells was examined by the CCK-8 assay. (C) The effect of RHPN2 on cell viability of lung adenocarcinoma was analyzed by colony formation assay. (C) The knockdown of RHPN2 significantly inhibited the tumor growth of subcutaneous tumors in nude mice. (D) The knockdown of RHPN2 significantly inhibited the weight of subcutaneous tumors in nude mice. (E) Lung adenocarcinoma tissues were confirmed by H\u0026amp;E staining. (F) The knockdown of RHPN2 significantly inhibited the expression of Ki-67 in subcutaneous tumor tissues of nude mice by IHC assay. * \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01, ** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755394/v1/31466681810156ada195ff6f.jpg"},{"id":84531887,"identity":"bf00cbec-30d6-4029-acf2-cfded20eb8d4","added_by":"auto","created_at":"2025-06-13 06:19:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5057124,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of RHPN2 on the metastasis of lung adenocarcinoma was investigated through \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e experiments. \u003c/strong\u003e(A) The effect of RHPN2 on the cell invasion and metastasis of lung adenocarcinoma cells was analyzed by transwell assay. (B) The effect of RHPN2 on the cell metastasis ability of lung adenocarcinoma cells was investigated\u003cstrong\u003e \u003c/strong\u003ethrough wound healing experiment. (C) Enrichment analysis based on TCGA data showed that the expression of RHPN2 was significantly positively correlated with the EMT pathway. (D) In lung adenocarcinoma cell lines, western blot was used to verify the effect of RHPN2 knockdown on the expression of proteins related to the EMT pathway. (E) One week and five weeks after tail veins were injected with A549 cells, the bioluminescence image of lung metastases were displayed. (F) The lung tissues of mice were dissected and removed, then the number of metastatic nodules on their surface was observed. (E) Lung metastasis tumors were confirmed via H\u0026amp;E staining. * \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01, ** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755394/v1/71782b42726d8e4a5e7152ca.jpg"},{"id":84531877,"identity":"c8865ebd-2ad6-40e1-b61d-194fa1828b19","added_by":"auto","created_at":"2025-06-13 06:19:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1367824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKLF5 increased \u003c/strong\u003e\u003cdel\u003e\u003cstrong\u003eup-regulates\u003c/strong\u003e\u003c/del\u003e\u003cstrong\u003e the expression of RHPN2 in lung adenocarcinoma through transcription. \u003c/strong\u003e(A) The effects of KLF5 knockdown or overexpression on the expression of RHPN2 mRNA in lung adenocarcinoma cells were verified respectively by RT-qPCR. (B) The effects of KLF5 knockdown or overexpression on the expression of RHPN2 protein in lung adenocarcinoma cells were verified respectively by western blot, respectively. (C) The subcellular localization of KLF5 and RHPN2 proteins in lung adenocarcinoma cells was observed through IF. (D) Motif of the KLF5 binding sites in the \u003cem\u003eRHPN2\u003c/em\u003e promoters predicted by the JASPAR dataset. (E) Schematic of the KLF5 binding sites on the \u003cem\u003eRHPN2\u003c/em\u003e promoters. (F) In the lung adenocarcinoma A549 cell line, the direct binding effect between KLF5 and site 1 of the \u003cem\u003eRHPN2\u003c/em\u003e promoters was confirmed by ChIP-qPCR. (G) The dual-luciferase reporter gene assay analysis revealed that KLF5 had an activating effect on RHPN2 in the lung adenocarcinoma A549 cell line. * \u003cem\u003eP \u003c/em\u003e\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755394/v1/dda2fec16df8f621f0361c2d.jpg"},{"id":84531884,"identity":"cae0ed5c-5262-4336-b7a9-17ce1abcde8e","added_by":"auto","created_at":"2025-06-13 06:19:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3172497,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRHPN2 facilitated the cell growth of lung adenocarcinoma in a KLF5-dependent form.\u003c/strong\u003e RHPN2 was overexpressed in KLF5-knockdown A549 cells, then the changes on cell proliferation were detected by CCK-8 (A) and colony formation (B) assays. Transplanted xenografts derived from A549 cells with sh-NC, sh-KLF5, and sh-KLF5+oeRHPN2 were established in nude mice (C). Tumor volume (C) and weight (D) were measured. (E) Lung adenocarcinoma tissues were histologically confirmed by H\u0026amp;E staining. (F) The expression changes of Ki-67 were detected by IHC assay. * \u003cem\u003eP \u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755394/v1/64f907ed3557aa281eda7353.jpg"},{"id":84532821,"identity":"667afa7f-d158-4125-8ac5-6a28147403c5","added_by":"auto","created_at":"2025-06-13 06:27:40","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4120778,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRHPN2 promoted the cell metastasis of lung adenocarcinoma in a KLF5-dependent form.\u003c/strong\u003e RHPN2 was overexpressed in KLF5-knockdown A549 cells, then the changes on cell invasion and migration ability were detected by transwell (A) and wound healing (B) assays. (C) Western blot analysis of EMT-related proteins (Snail, E-cadherin, N-cadherin, Vimentin) in A549 cells. (D) Bioluminescence imaging of nude mice bearing lung adenocarcinoma metastasis at week 1 and week 5 after tail vein injection of A549 cells transfected with sh-NC, sh-KLF5, or sh-KLF5+ oeRHPN2. (E) Representative images of metastatic nodules on the surface of lung tissues in nude mice. (F) H\u0026amp;E staining of tumor metastatic nodules in the lung tissues of nude mice and statistical analysis of the number of nodules on the surface of the lung tissues. * \u003cem\u003eP \u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755394/v1/ba1887f375c07cb934fd6845.jpg"},{"id":84531890,"identity":"6ba9bca8-aff9-4573-8ebc-f013c0549ec6","added_by":"auto","created_at":"2025-06-13 06:19:40","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3348363,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKLF5 expression was positively correlated with RHPN2 in lung adenocarcinoma. \u003c/strong\u003e(A) The expression of KLF5 and RHPN2 proteins in lung adenocarcinoma tissues was detected by IHC. (B) The expression of KLF5 was positively correlated with that of RHPN2 in 20 cases of lung adenocarcinoma tissues. (C) Schematic diagram of KLF5 facilitating lung adenocarcinoma metastasis via regulating the EMT pathway through RHPN2.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755394/v1/16a8c84057958004687577a5.jpg"},{"id":93419634,"identity":"3ddbea0e-8d81-4a7c-8c7e-fd0ba8ccfc33","added_by":"auto","created_at":"2025-10-13 16:04:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28410785,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6755394/v1/0e47e661-438a-4c3a-8d76-d439f8830ee9.pdf"},{"id":84532818,"identity":"9bd6cfc3-9cf8-4622-8d12-0f2da3e3dcad","added_by":"auto","created_at":"2025-06-13 06:27:40","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":15273,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6755394/v1/8449f49e233e5fdb509badd4.docx"}],"financialInterests":"","formattedTitle":"KLF5 facilitates lung adenocarcinoma metastasis via regulating the epithelial-mesenchymal transition pathway through RHPN2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIt was estimated 1,060,600 new lung cancer cases, accounting for 22.0% of all malignancies, and around 733,300 new lung cancer deaths, accounting for 28.5% of all deaths from malignant tumors, occurred in China in 2022\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Lung adenocarcinoma, characterized by insidious onset and early distant metastasis, is the most common sub-type for lung cancer. Distant metastasis is the main reason reducing the survival time of patients with advanced lung adenocarcinoma. Despite significant progresses for the treatment of lung adenocarcinoma, the 5-year survival rate to advanced patients is still less than 20%\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The mechanism of distant metastasis of lung adenocarcinoma cells is not entirely understood. It is well known that metastasis of tumor cells, including lung adenocarcinoma, is a complex multi-step process involving changes in multi-dimensional network systems, for instance aberrant changes of genome, transcriptome, proteomics and metabolites\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our prior research, the transcription factor Kruppel-like factor 5 (KLF5) was reported to facilitate the malignant progression of lung adenocarcinoma through its direct interaction with ubiquitin-specific peptidase 38\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. In a recent single-cell analysis of patients with both metastatic and non-metastatic tumors, researchers delved into the intricate regulatory network of transcription factors that are active at various stages of metastasis. Through this comprehensive investigation, KLF5 is identified as a pivotal regulator deeply involved in cancer metastasis progress\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.Through constructing regulatory networks for transcription factors on breast cancer through bulk, single-cell and single-nucleus multi-omic techniques as well as spatial transcriptomics and multiplex imaging, the importance of KLF5 in basal-like tumors and luminal progenitors are elaborately investigated\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. In addition, KLF5 is reported to function as a crucial role in mediating glutamine metabolism, thereby exerting a significant influence on tumor cell growth in non-small cell lung cancer\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Furthermore, KLF5 has been identified as a critical regulator involved in chemokine production and neutrophil recruitment in lung squamous cell carcinoma, thereby significantly influencing the tumor immune microenvironment\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Taken all into account, the impact of KLF5 on lung adenocarcinoma is both comprehensive and highly significant. However, the downstream molecular mechanism of KLF5 in lung adenocarcinoma remains largely unclear.\u003c/p\u003e \u003cp\u003eIn the current study, we identified GTP-HO-binding Protein 2 (RHPN2) as the downstream target gene of KLF5 by bioinformatics methods. Combined bioinformation analysis and tissue sample detecting identification RHPN2 is found highly expressed in lung adenocarcinoma and is a critical factor contributing to the poor prognosis of patients. In vitro and in vivo experiments revealed that RHPN2 is able to promote the growth and metastasis of lung adenocarcinoma cells. More importantly, we demonstrated that RHPN2 promotes the process of epithelial mesenchymal transformation (EMT) in lung adenocarcinoma cells in a KLF5-dependent form.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eChromatin-immunoprecipitation (ChIP)\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e-seq data analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe KLF5 ChIP-seq data used in this study are available in the GENE EXPRESSION OMNIBUS (GEO) database under accession number GSE164852\u003csup\u003e[9]\u003c/sup\u003e.\u0026nbsp;Quality control of the raw FASTQ data was performed using FastQC (v0.11.9)\u003csup\u003e[10]\u003c/sup\u003e to assess data quality. Trimmomatic (v0.39)\u003csup\u003e[11]\u003c/sup\u003e was used to trim low-quality bases and adapter sequences, and reads shorter than 36 bp were filtered out. The clean reads were aligned to the hg19 reference genome using Bowtie2 (v2.4.4)\u003csup\u003e[12]\u003c/sup\u003e. The resulting SAM files were converted to BAM format using SAMtools (v1.12)\u003csup\u003e[13]\u003c/sup\u003e, and duplicate reads were removed using Picard tools (v2.25.0)\u003csup\u003e[14]\u003c/sup\u003e to reduce PCR amplification bias. Peak calling was performed using MACS2 (v2.2.7.1)\u003csup\u003e[15]\u003c/sup\u003e with the parameters -B, --qvalue 0.01, and --gsize hs to identify genomic binding sites. The output BED files contained peak locations and significance scores. Peaks were annotated using ChIPseeker (v1.30.3)\u003csup\u003e[16]\u003c/sup\u003e to analyze their distribution across genomic features (e.g., promoter regions, exons) and to associate them with the nearest genes using the TxDb.Hsapiens.UCSC.hg19.knownGene database. Peak distribution statistics were generated, and the data were visualized using the Integrative Genomics Viewer (IGV, v2.11.1)\u003csup\u003e[17]\u003c/sup\u003eto display binding peaks in the context of genomic annotations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)\u0026nbsp;enrichment analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGO and KEGG pathway enrichment analyses were performed using clusterProfiler (v4.2.2)\u003csup\u003e[18]\u003c/sup\u003e. Target genes were extracted based on the nearest genes to the peaks identified by ChIPseeker. For GO analysis, enrichment was performed for Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) categories, with a significance threshold of \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 and Benjamini-Hochberg correction for multiple testing. For KEGG analysis, target genes were mapped to the KEGG pathway database, and significantly enriched pathways (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) were identified.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe Cancer Genome Atlas Program (TCGA)\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data on lung adenocarcinoma in the TCGA database (https://portal.gdc.cancer.gov) were used to analyze the expression of RHPN2 and its relationship with the prognosis of patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTissue samples and cell lines\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty tumor tissues and their corresponding acjacent normal tissues were collected from patients with lung adenocarcinoma to detect of RHPN2 expression. These patients were diagnosed with lung adenocarcinoma through pathology and received surgical treatment at The First Hospital of Lanzhou University from March to June 2020. They did not receive radiotherapy, chemotherapy or immunotherapy before the operation. All tissues were immediately frozen in liquid nitrogen after resection and then stored at -80\u0026deg;C. This study was approved by the Ethics Committee of The First Hospital of Lanzhou University in China (LDYYLL2022-448).\u003c/p\u003e\n\u003cp\u003eHuman normal lung epithelial cell line (BEAS-2B) and lung adenocarcinoma cell line (A549, H1299, H1975 and HCC827) were derived from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, NY, USA), which contained 1% penicillin/streptomycin (Invitrogen, CA, USA) and 10% fetal bovine serum (FBS; Gibco) added 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C, humidified incubator (Thermo Scientific, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHematoxylin and Eosin (H\u0026amp;E) staining and Immunohistochemical (IHC) staining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tissues were fixed with a 10% formaldehyde solution and then dehydrated with gradient ethanol. Briefly, the tissues were embedded with paraffin wax and then made into 4 \u0026mu;m continuous sections. First, immerse the slices in the hematoxylin staining solution for 3 to 5 minutes. After removing the excess hematoxylin with 1% hydrochloric acid alcohol, immerse the slices in eosin staining solution for 1-2 minutes. Dehydration is carried out again using gradient ethanol. Finally, use neutral gum to seal the slices.\u003c/p\u003e\n\u003cp\u003eIHC staining was performed on the prepared tissue sections. The sections were soaked in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 5 minutes at room temperature to remove endogenous peroxidase activity, then incubated in sodium citrate buffer at 95\u0026deg;C for 15 minutes to repair the antigen. The sections were blocked by 5% normal goat serum for 10 minutes to seal the non-specific binding site. Next, the indicated primary antibody was added and incubated at 4℃ overnight. Finally, the secondary antibody labeled with horseradish peroxidase was incubated for 1 hour. The IHC staining results were evaluated by combining the staining intensity and the proportion of tumor cells. The final immune response score was calculated as the staining intensity score \u0026times; the percentage of positive cells. The immune response score of 0-4 indicates low expression, and 5-9 indicates high expression. The antibodies used in this study are listed in \u003cstrong\u003eTable S1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLentivirus transduction\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSmall hairpin RNA (shRNA) targeting KLF5 and RHPN2, and containing amplified human RHPN2 or KLF5 sequence vectors were synthesized by HANBIO Co., Ltd. (Shanghai, China) and packaged as lentivirus particles. Then, lentivirus was transfected into lung adenocarcinoma cells, and puromycin was used to screen the stably transfected cell lines. The shRNA sequences are listed in \u003cstrong\u003eTable S2\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWestern blot\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins in cells or tissues were extracted using radioimmunoprecipitation assay buffer (RIPA, Beyotime, Shanghai, China), and quantified using the biuretic acid assay kit (BCA, Beyotime) in accordance with the manufacturer\u0026apos;s instructions. The protein was transferred onto the polyvinylidene fluoride (PVDF) membrane (Millipore, Boston, MA, USA) and incubated overnight with the primary antibody targeting the target protein at 4\u0026deg;C. The next day, the membrane was incubated with the indicated secondary antibody at room temperature for 1 hour. The immunoreactivity was observed using the automatic chemiluminescence image processing system (Bio-Rad, San Jose, CA, USA). The antibodies used in this study are listed in \u003cstrong\u003eTable S1\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell viability assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell counting kit 8 (CCK8, Yelasen, Shanghai, China) and colony formation assay was used to detect the cell viability of lung adenocarcinoma cells, which was performed as described previously\u003csup\u003e[4]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTranswell assay and wound healing assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transwell assay and wound healing assay were used to evaluate the cell migration ability of lung adenocarcinoma cells, which was performed as described previously\u003csup\u003e[19]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRT-qPCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-qPCR was performed as described previously\u003csup\u003e[19]\u003c/sup\u003e. The primer sequences are listed in \u003cstrong\u003eTable S3\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunofluorescence assay (IF)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence assay was performed as described previously\u003csup\u003e[11]\u003c/sup\u003e. The antibodies used in this study are listed in \u003cstrong\u003eTable S1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eJASPAR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe JASPAR database (https://jaspar.genereg.net/)\u003csup\u003e[20]\u003c/sup\u003e was used to identify the binding site of transcription factor KLF5 to the RHPN2 promoter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eChIP-qPCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the manufacturer\u0026apos;s instructions, ChIP assay was conducted using the SimpleChIP\u0026reg; Enzymatic Chromatin IP Kit (CST, Massachusetts, MA, USA). In detail, A549 cells were fixed with 1% paraformaldehyde for 15 minutes, then 0.125M glycine was added and quenched for 10 minutes. A lysis buffer containing protease inhibitors was added to the cells, and chromatin fragments were generated using an ultrasonic fragmentation device. The cleaved chromatin was incubated overnight with KLF5 antibody (Proteintech, Wuhan, China) and Protein G magnetic beads at 4\u0026deg;C. Rabbit IgG (CST, Massachusetts, MA, USA) was served as a negative control. After washing and elution, the purified DNA was used for qPCR analysis. The primers sequences for ChIP-qPCR are listed in \u003cstrong\u003eTable S4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLuciferase activity assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLuciferase activity assay was performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer\u0026apos;s instructions. Briefly, wild-type (WT) and mutant (Mut1) RHPN2 promoter sequences were amplified and cloned into the pGL4.10-Basic vector (Promega), generating constructs named pGL4.10-RHPN2-promoter and pGL4.10-RHPN2-Mut1-promoter, respectively. The A549 cell line was co-transfected with these promoter constructs along with either pcDNA3.1-KLF5 expression plasmid or control pcDNA3.1 vector using Lipofectamine 3000 (Invitrogen, USA). Cells were cultured for 48 hours post-transfection. Luciferase activity was measured using a luminometer (GloMax, Promega), and results were normalized to Renilla luciferase activity as an internal control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimal model\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eweek-old male BALB/c nude mice (Ziyuan Laboratory of Animal Science and Technology, Hangzhou, China) were used to construct animal models. Nude mice were randomly divided into the following 4 groups (n=5 each): sh-NC, sh-RHPN2#3, sh-KLF5, and sh-KLF5+oeRHPN2. The subcutaneous tumorigenesis model of nude mice was used to simulate the growth of tumors \u003cem\u003ein vivo\u003c/em\u003e. 4\u0026times;10\u003csup\u003e6\u003c/sup\u003e A549 cells were inoculated subcutaneously into each mouse, and the volume of the tumor was measured weekly using a vernier caliper. The calculation formula for tumor volume is as follows: volume = (length \u0026times; width\u003csup\u003e2\u003c/sup\u003e)/2. The mice were euthanized after four weeks and the tumor tissue was collected and weighed. Then the tumor tissues were immobilized with formalin for subsequent analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAn animal model of tumor metastasis i\u003cem\u003en vivo\u003c/em\u003e was simulated by injecting 3\u0026times;10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003eA549-luc cells through the tail vein of nude mice. The bioluminescence images were acquired by the IVIS spectrum imaging system (PerkinElmer, Massachusetts, USA). Five weeks later, the mice were euthanized and their lung tissues were dissected. Then, the number of metastatic nodules on the surface of the lung tissues was calculated. Animal experiments were reviewed and approved by the Ethics Committee of The First Hospital of Lanzhou University. All animals were cared for in accordance with the Guide for the \u0026ldquo;Care and Use of Laboratory Animals\u0026rdquo;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eContinuous variables were expressed as mean \u0026plusmn; standard deviation. Differences between two groups were assessed using the independent samples \u003cem\u003et\u003c/em\u003e-test. For comparisons among three or more groups, one-way analysis of variance (ANOVA) was employed, followed by the least significant difference (LSD) test for pairwise comparisons. The correlation between continuous variables was assessed using Pearson\u0026apos;s correlation analysis, with R representing the correlation coefficient. All statistical analyses and graphical representations were performed using GraphPad Prism version 8.0 and R software (version 4.4.1). \u003cem\u003eP\u003c/em\u003e value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eKLF5 ChIP-seq analysis in lung adenocarcinoma cells reveals key regulatory roles\u003c/h2\u003e \u003cp\u003eThe genomic binding profile of KLF5, a transcription factor implicated in lung adenocarcinoma, was investigated through ChIP-seq analysis. The distribution of KLF5 binding peaks shows a higher-level enrichment at promoter regions and distal intergenic regions, consistent with its role as a transcription factor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This observation is further supported by the concentration of binding peaks within \u0026plusmn;\u0026thinsp;0.5 kb of transcription start sites (TSS), indicating that KLF5 primarily regulates gene expression by binding near promoter regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Functional annotation of KLF5 binding peak-annotated genes through GO and KEGG pathway analysis provides insights into the biological processes and pathways regulated by KLF5 in lung adenocarcinoma cells. The BP analysis highlights KLF5's involvement in critical cellular functions such as cell migration (ameboidal-type, endothelial, and epithelial), epithelial cell proliferation, and tissue morphogenesis, processes closely linked to cancer progression and metastasis. The CC analysis reveals associations with key cellular structures, including adherens junctions, focal adhesions, and membrane microdomains, which are essential for cell-cell communication and signaling. The MF analysis further supports KLF5's role in transcriptional regulation, with enriched terms such as DNA-binding transcription activator activity and RNA polymerase II-specific binding. Additionally, KEGG pathway analysis identifies significant enrichment in pathways related to cancer progression, including the Hippo signaling pathway, Rap1 signaling pathway, and small cell lung cancer, suggesting that KLF5 may play a essential role on modulating these oncogenic pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong those genes with higher KLF5 binding signals, five (ADAP1, CTNNA1, DUSP26, MYH14, and RHPN2) are highlighted as potential direct transcriptional targets of KLF5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Notably, RHPN2 shows a significant positive correlation with KLF5 expression, with a correlation coefficient of 0.38 and a \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001, suggesting a potential regulatory relationship between KLF5 and RHPN2 in lung adenocarcinoma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Collectively, these findings emphasize KLF5's central role on regulating genes and pathways associated with lung adenocarcinoma progression, particularly in processes related to cell migration, proliferation, and other oncogenic signaling pathways. The identification of KLF5 binding peak-annotated genes and their functional annotations provide a foundation for further exploration of KLF5's mechanistic contributions to lung adenocarcinoma biology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eRHPN2 is highly expressed in lung adenocarcinoma\u003c/h2\u003e \u003cp\u003eAs previously mentioned, RHPN2 is a potential target gene for the transcription factor KLF5, while its role on the progression of lung adenocarcinoma is unclear. Based on the data analysis for lung adenocarcinoma in the TCGA database, RHPN2 expression was found up-regulated in lung adenocarcinoma samples compared to normal samples \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Moreover, High RHPN2 expression is associated with shorter survival in patients with lung adenocarcinoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Univariate and multivariate analysis suggested that high expression of RHPN2 was an independent risk factor for poor prognosis of lung adenocarcinoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). Next, we detected the expression of RHPN2 in lung adenocarcinoma cell lines and tissues by western blot and IHC, respectively. Compared with human bronchial epithelial BEAS-2B cell line, RHPN2 expression was significantly up-regulated in lung adenocarcinoma cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Among them, the A549, H1299, and H1975 cell lines were used in the subsequent experiments. In addition, the expression level of RHPN2 in lung adenocarcinoma tissues was significantly higher than that in adjacent tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). At the same time, RHPN2 protein was mainly located in the cytoplasm of lung adenocarcinoma cells through IHC detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). In summary, RHPN2 is highly expressed in lung adenocarcinoma and is an independent risk factor for predicting poor prognosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eRHPN2 promotes the growth of lung adenocarcinoma\u003c/h2\u003e \u003cp\u003eTo explore the effect of RHPN2 on the progression of lung adenocarcinoma, we first constructed a RHPN2-deficient lung adenocarcinoma cell lines. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the inhibition rates in RHPN2 lung adenocarcinoma cell lines A549 and H1299 were verified by western blot. Based on the western blot results, sh-RHPN2#2 and #3 showed better inhibitory effects and were therefore used in subsequent functional studies. Next, we focused on investigating the effect of RHPN2 on the cell proliferation of lung adenocarcinoma cells through the CCK-8 and colony formation assays. The results of CCK-8 assay showed that knockdown of RHPN2 could lead to a significant decrease in the cell viability of A549 and H1299 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The colony formation experiments also showed similar trends and results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Meanwhile, we constructed a subcutaneous tumor model in nude mice using the A549 cell line (sh-RHPN2#3) to verify the effect of RHPN2 on the growth of lung adenocarcinoma \u003cem\u003ein vivo\u003c/em\u003e. Compared with the control group, the volume and weight of subcutaneous xenografts in nude mice in the sh-RHPN2#3 group were significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). Histological analysis by H\u0026amp;E staining confirmed that the xenografts were indeed tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Finally, we detected the expression changes of the cell proliferation marker Ki-67 protein in the xenograft tissues through IHC, and found that knockdown of RHPN2 could inhibit its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Overall, RHPN2 may promote the cell proliferation \u003cem\u003ein vitro\u003c/em\u003e and tumor growth \u003cem\u003ein vivo\u003c/em\u003e for lung adenocarcinoma.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eRHPN2 facilitates tumor metastasis of lung adenocarcinoma\u003c/h2\u003e \u003cp\u003eGiven the clinical association of RHPN2 with tumor metastasis of lung adenocarcinoma, we then focused on studying its role in regulating the migration and invasion of lung adenocarcinoma cells. Transwell and wound healing assays showed that knockdown of RHPN2 significantly inhibited the cell migration and invasion of A549 and H1299 cell lines, highlighting its critical role in lung adenocarcinoma metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). Then, we analyzed the potential molecular biological mechanism of RHPN2 in the process of lung adenocarcinoma through Gene Set Enrichment Analysis (GSEA). The GSEA results indicated that RHPN2 was closely associated with multiple tumor-related pathways, including poor survival of lung cancer and EMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Among them, EMT is an important precursor step for distant metastasis of cancer cells. Therefore, we analyzed the effect of RHPN2 on the expression of EMT-related proteins in lung adenocarcinoma cell lines by western blot. The results showed that knockdown of RHPN2 could significantly inhibit the expressions of Snail, N-cadherin, and Vimentin, but up-regulated the expression level of E-cadherin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Those results increase the favorable evidence that RHPN2 could promote the cell migration of lung adenocarcinoma cells. Finally, we constructed an animal model of \u003cem\u003ein vivo\u003c/em\u003e metastasis of tumor cells by injecting lung adenocarcinoma cells into the tail vein of nude mice, and the successful modeling was proved by using fluorescence imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Five weeks after injecting A549-luc cells into the tail vein of mice, we dissected their lungs and calculated the number of nodules on the lung surface, and confirmed that they were metastatic tissues through H\u0026amp;E staining. The results showed that knockdown of RHPN2 could significantly repress the metastasis of lung adenocarcinoma in nude mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H). The above-mentioned studies have proved that RHPN2 may facilitate the distant metastasis of lung adenocarcinoma.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eKLF5 transcriptionally upregulates RHPN2 expression in lung adenocarcinoma\u003c/h2\u003e \u003cp\u003eIn previous studies, we have confirmed that RHPN2 is upregulated in lung adenocarcinoma and promotes the malignant phenotype of tumor cells. Next, we focused on investigating whether the high expression of RHPN2 was regulated by the transcription factor KLF5. RT-qPCR and western blot confirmed that knockdown of KLF5 could inhibit the expression level of RHPN2 in A549 cells, respectively, while overexpression of KLF5 further promoted the expression level of RHPN2 in H1975 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). Those results indicate that KLF5 has a positive regulatory effect on RHPN2 in lung adenocarcinoma. Then, we detected the localization of KLF5 and RHPN2 proteins in lung adenocarcinoma cells through IF staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, the KLF5 protein was mainly located in the cell nucleus, and the RHPN2 protein was mainly located in the cytoplasm of lung adenocarcinoma cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate whether the KLF5 protein could directly bind to the promoter regions of \u003cem\u003eRHPN2 gene\u003c/em\u003e, we first used the JASPAR database to determine three sites where the \u003cem\u003eRHPN2\u003c/em\u003e\u0026rsquo;s promoter region may bind to KLF5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E). We precisely detected the binding sites on those \u003cem\u003eRHPN2\u003c/em\u003e promoters by ChIP-qPCR. We detected the direct action of KLF5 protein on the binding sites of these \u003cem\u003eRHPN2\u003c/em\u003e promote regions in A549 cells by ChIP-qPCR. Results showed that the second site (-686 to -697) in the promoter regions of \u003cem\u003eRHPN2\u003c/em\u003e was an important binding site for the KLF5 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Subsequently, in order to further verify the transcriptional activation effect of the transcription factor KLF5 on RHPN2, we conducted the dual-luciferase reporter gene assay using A549 cells. Based on the ChIP-qPCR results, we mutated the first site where the KLF5 protein mainly binds in the \u003cem\u003eRHPN2\u003c/em\u003e promoter region, and then detected the activity of luciferase. The luciferase activity of the wild-type \u003cem\u003eRHPN2\u003c/em\u003e promoter was significantly higher than that of the mutant \u003cem\u003eRHPN2\u003c/em\u003e promoters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). These results indicate that there is a positive regulatory relationship between the transcription factor KLF5 and its downstream target gene RHPN2. In conclusion, KLF5 promotes the expression of RHPN2 in lung adenocarcinoma through transcriptional upregulation.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eRHPN2 promotes the progression of lung adenocarcinoma in a KLF5-dependent form\u003c/h2\u003e \u003cp\u003eTo further clarify that RHPN2 promotes the process of lung adenocarcinoma in a KLF5-dependent form, we conducted rescue experiments by overexpress RHPN2 in sh-KLF5 A549 cells and then examined the changes in cell proliferation and migration of lung adenocarcinoma. CCK8 and colony formation assays demonstrated that KLF5 knockdown inhibited the cell proliferation of A549 cells, meanwhile, overexpression of RHPN2 reversed part of this trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). These results indicated that RHPN2 overexpression reversed the inhibitory role of KLF5 knockdown on tumor cell growth of lung adenocarcinoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D). Similarly, we performed IHC staining of Ki-67 on the tumor tissues. Results showed that knockdown of KLF5 inhibited the expression of Ki-67, while overexpression of RHPN2 reversed this trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F). Additionally, KLF5 knockdown inhibited the cell migration and invasion capacity of A549 cells, and the sh-KLF5-induced reduction of trend was partially rescued by co-transfection with RHPN2 in transwell and wound healing assays, (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B). Western blot revealed that the sh-KLF5-induced decrease in Snail, N-cadherin, and Vimentin partially reversed by the overexpression of RHPN2, and the sh-KLF5-induced increase in E-cadherin expression was also partially reversed by RHPN2 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). In the nude mouse model of lung adenocarcinoma metastasis \u003cem\u003ein vivo\u003c/em\u003e, we observed that knockdown of KLF5 significantly reduced the number of lung metastatic nodules, while RHPN2 was found to reverse the inhibitory effect of sh-KLF5 on metastatic nodules (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-F). Collectively, \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies have shown that RHPN2 promotes the growth and metastasis of lung adenocarcinoma in a form dependent on the transcription factor KLF5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eKLF5 expression is positively correlated with RHPN2 in lung adenocarcinoma\u003c/h2\u003e \u003cp\u003eTo further clarify the correlation between KLF5 and RHPN2 in lung adenocarcinoma, we detected the expression of both KLF5 and RHPN2 in 20 cases of lung adenocarcinoma tissues through IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). According to the scoring criteria of IHC, we statistically found that the expression level of KLF5 was significantly positively correlated with RHPN2 in lung adenocarcinoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Those results strengthen the evidence that RHPN2 is a key target of the transcription factor KLF5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLung adenocarcinoma has an insidious onset and is characterized by distant metastasis at an early stage. Distant metastasis is an important factor contributing to a poor prognosis in patients with lung adenocarcinoma. The molecular mechanism of distant metastasis of lung adenocarcinoma remains unclear, and in-depth exploration of it will help improve the poor prognosis of patients. The EMT program, as a set of multiple and dynamic transition states between epithelial and mesenchymal phenotypes, has been clarified as an important precursor step for distant metastasis of cancer cells\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Multiple studies have confirmed the key role of the EMT pathway in the progression of lung adenocarcinoma. It is reported that CHIP-mediated CIB1 ubiquitination regulated EMT and tumor metastasis in lung adenocarcinoma\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In addition, cigarette smoke was identified to induce EMT, stemness, and metastasis in lung adenocarcinoma cells via upregulated RUNX-2/galectin-3 pathway\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In the present study, we first confirmed that RHPN2 was upregulated in lung adenocarcinoma and was closely associated with the activation of the EMT pathway.\u003c/p\u003e \u003cp\u003eRHPN2 is a Rho GTPase-binding protein, which was initially discovered to be involved in the recombination of the cytoskeleton and the alteration of cell morphology \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Recent studies have shown that RHPN2 plays an important role on oncogenic signaling in various tumors, including promoting cell proliferation, migration and invasion of tumor cells\u003csup\u003e[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Up to date, little is known for RHPN2 in lung adenocarcinoma. He et al. discovered that RHPN2 is necessary for cell proliferation and invasion of lung cancer cells. Interestingly, overexpression of RHPN2 endows lung cancer cells with resistance to glutamine depletion\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In the current study, we systematically analyzed the role of RHPN2 in lung adenocarcinoma through multiple methods. Based on bioinformatics analysis and tissue sample confirming, we found that RHPN2 was upregulated in lung adenocarcinoma and was closely associated with the poor prognosis of patients. Moreover, \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments confirmed that RHPN2 could promote the growth and metastasis of lung adenocarcinoma. More importantly, our research clarified that RHPN2 promotes the metastasis of lung adenocarcinoma cells by regulating the EMT pathway. The above results highlight the importance of RHPN2 in the process of lung adenocarcinoma. However, the specific mechanism of RHPN2 in lung adenocarcinoma remains unclear.\u003c/p\u003e \u003cp\u003eHerein, we identified that RHPN2 interacts with KLF5 to exert its oncogenic functions lung adenocarcinoma. Multiple studies have shown that KLF5 plays an important role in the progression of lung adenocarcinoma\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e][\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Our previous research found that the N-terminal of USP38 (residues 1-400aa) interacts with the residues 1-200 aa of KLF5, thereby stabilizing the KLF5 protein through deubiquitination\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Our analysis revealed that KLF5, as a transcription factor, could bind to different gene promoter regions through bioinformatics. Among them, RHPN2 is the target gene of KLF5 and is closely related to it in lung adenocarcinoma. Mechanically, we clarified that KLF5 affects the expression of RHPN2 in lung adenocarcinoma through transcriptional activation. Further rescue experiment confirmed that RHPN2 promotes the progression of lung adenocarcinoma in a KLF5-dependent manner, especially in terms of the regulation of the EMT pathway. These findings suggest that targeting the KLF5/RHPN2/EMT axis may be a promising therapeutic strategy for lung adenocarcinoma. Future research could explore small molecule inhibitors targeting the KLF5/RHPN2 axis to test their indirect regulation of the EMT pathway. This study also has certain limitations. For example, the exploration of the regulatory mechanisms of KLF5 and RHPN2 is relatively single, and it is debatable whether there are complexes or super enhancers affecting their interaction. In addition, the cell or animal models we selected are relatively simple. Future research could verify the effectiveness of these strategies under the condition of simulating the tumor microenvironment by using organoid models (PDO) and patient-derived xenotransplantation (PDX) models, thereby accelerating their clinical transformation process.\u003c/p\u003e \u003cp\u003eThis study revealed the regulatory role of RHPN2 in the growth and metastasis of lung adenocarcinoma, emphasizing that it regulates the EMT pathway in a KLF5-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). This novel finding not only enhances our understanding of EMT signaling, but also provides new insights into the potential of targeting this pathway in the treatment of lung adenocarcinoma.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eKLF5, transcription factor Kruppel-like factor 5; RHPN2, GTP-HO-binding Protein 2; \u0026nbsp;EMT, epithelial mesenchymal transformation; GEO, GENE EXPRESSION OMNIBUS; ChIP, Chromatin-immunoprecipitation; IGV, Integrative Genomics Viewer; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BP, Biological Process; MF, Molecular Function; CC, Cellular Component; TCGA, The Cancer Genome Atlas Program; RPMI, Roswell Park Memorial Institute; FBS, fetal bovine serum; H\u0026amp;E, Hematoxylin and Eosin; IHC, Immunohistochemical; shRNA, small hairpin RNA; RIPA, radioimmunoprecipitation assay buffer; BCA, biuretic acid assay; PVDF, polyvinylidene fluoride; CCK8, cell counting kit 8; IF, immunofluorescence assay; WT, wild-type; Mut, mutant; ANOVA, one-way analysis of variance; LSD, least significant difference; TSS, transcription start sites; GSEA, Gene Set Enrichment Analysis; PDO, organoid models; PDX, patient-derived xenotransplantation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Professor Ran-ran sun for his guidance and assistance in animal experiments. ChIP-qPCR service was provided by Shanghai Cutseq Biomedical Technology Co., Ltd. We also acknowledge TCGA, GEO and JASPAR database for providing their platforms and contributors for uploading their meaningful datasets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTZ, RQW and YBY drafted the article and interpreted data. TZ, RQW, YBY, WJZ, ZQD and XLM performed the experiments and obtained the data. YBY and QS conducted bioinformatic analysis. JJG and QDZ collected clinical samples. XYS assisted in revising the format of the manuscript. YBL, FS, and XMH designed the study and revised the article critically for important intellectual content. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the (1) Youth Science and Technology Talent Innovation Project of Lanzhou City (2023-QN-14), (2) Medical Innovation and Development Project of Lanzhou University (lzuyxcx-2022-183), (3) Science and Technology Plan Project in Chengguan District of Lanzhou City (2021RCCX0008), (4) Key Research and Development Plan from Gansu Provincial Department of Science and Technology (22YF7FA086), (5) Unite Pesearch Foundation of Gansu Province (23JRRA1497), \u0026nbsp;(6) Natural Science Foundation of Gansu Province (23JRRA0928 and 23JRRA1607), (7) Key Talent Project of Gansu Province (2025RCXM036).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHan B, Zheng R, Zeng H, Wang S, Sun K, Chen R, et al. 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Oncogenesis. 2023;12(1):35.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"lung adenocarcinoma, RHPN2, KLF5, EMT, metastasis, transcription","lastPublishedDoi":"10.21203/rs.3.rs-6755394/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6755394/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eBackground\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDistant metastasis is the primary reason shortening the survival time for patients with advanced lung adenocarcinoma. Transcription factor Kruppel-like factor 5 (KLF5) has been confirmed to facilitate the progression of lung adenocarcinoma. However, the certain mechanism by which KLF5 involves in the tumor metastasis of lung adenocarcinoma remains largely unclear.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethods\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGpt-ho-binding Protein 2 (RHPN2) was screened as the potential downstream target gene for KLF5 via bioinformatics analysis, which closely participates in regulation of the epithelial mesenchymal transformation (EMT) pathway in lung adenocarcinoma. Western blot and immunohistochemistry assays were performed to examine RHPN2 expression in lung adenocarcinoma. \u003cem\u003eIn vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments were conducted to explore the regulatory role of RHPN2 on cell growth and metastasis of lung adenocarcinoma. Chromatin immunoprecipitation sequencing (ChIP-seq) was used to analyze the direct binding activity between the KLF5 and \u003cem\u003eRHPN2\u003c/em\u003e promoter regions. The luciferase activity assay was carried out to verify the transcriptional activation effect of KLF5 to RHPH2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRHPN2 was found to be highly expressed in lung adenocarcinoma and patients with highly RHPN2 expression showed a poor prognosis in lung adenocarcinoma. \u003cem\u003eIn vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e, experiments identified that RHPN2 promoted the cell growth and metastasis, and activated the EMT pathway in lung adenocarcinoma. Machanismly, KLF5 could directly bind to the promoter regions of RHPN2 and up-regulate the expression of the latter in lung adenocarcinoma through translational activation. In addition, the rescue experiments confirmed that \u003cem\u003eRHPN2\u003c/em\u003e facilitated the progression of lung adenocarcinoma at least in a KLF5-dependent form.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusion\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur study offers insights into the potential mechanisms of metastasis in lung adenocarcinoma and suggests RHPN2 as a potential therapeutic target.\u003c/p\u003e","manuscriptTitle":"KLF5 facilitates lung adenocarcinoma metastasis via regulating the epithelial-mesenchymal transition pathway through RHPN2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-13 06:19:35","doi":"10.21203/rs.3.rs-6755394/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-06-10T22:53:36+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-10T22:40:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-28T15:36:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2025-05-27T01:02:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cf04a85d-a9c0-4cac-9072-8b3453a8c7e1","owner":[],"postedDate":"June 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T15:59:41+00:00","versionOfRecord":{"articleIdentity":"rs-6755394","link":"https://doi.org/10.1186/s12967-025-07150-6","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2025-10-10 15:57:12","publishedOnDateReadable":"October 10th, 2025"},"versionCreatedAt":"2025-06-13 06:19:35","video":"","vorDoi":"10.1186/s12967-025-07150-6","vorDoiUrl":"https://doi.org/10.1186/s12967-025-07150-6","workflowStages":[]},"version":"v1","identity":"rs-6755394","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6755394","identity":"rs-6755394","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}