The role of SH3RF2 in lung squamous cell carcinoma and M2 polarization: insights into LZTS2 ubiquitination

The role of SH3RF2 in lung squamous cell carcinoma and M2 polarization: insights into LZTS2 ubiquitination | 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 The role of SH3RF2 in lung squamous cell carcinoma and M2 polarization: insights into LZTS2 ubiquitination Jie Yang, Zhongfei Jia, Juan Li, Chao Jiang, Xin Zhao, Yuxiang Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5457209/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Jul, 2025 Read the published version in Biology Direct → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Although the treatment of lung cancer has been well developed, the survival rate for lung squamous cell carcinoma (LUSC) is still low. It is meaningful to explore new molecular targets and develop new treatment strategies. SH3RF2 is an E3 ubiquitin ligase containing 3 SH3 domains and has not been reported in LUSC. Results: SH3RF2 promoted the proliferation of LUSC cells, and the nuclear translocation of β-catenin, increased Arg-1, CD163 and IL-10 RNA levels and the proportion of CD206+ cells in M0 THP-1 cells, and enhanced the migration and invasion of M0 THP-1 cells. ICG-001 alleviated the above effects of SH3RF2 on M0 THP-1 cell. In vivo tumorigenesis experiments found that SH3RF2 promoted tumor growth and increased the proportion of M2 cells. IP found that SH3RF2 interacted with LZTS2 and regulated the ubiquitination of LZTS2 with RING domain. LZTS2 overexpression reduced the nuclear translocation of β-catenin, cell migration and invasion, and M2 polarization promoted by SH3RF2 overexpression. The combination of SH3RF2 overexpression and radiotherapy inhibited the growth of tumor. Conclusions: This study elucidates the cancer-promoting role of SH3RF2, its positive effect on M2 polarization, and its relationship with LZTS2 and β-catenin. It provides new candidate molecular targets for the treatment of LUSC. Lung squamous cell carcinoma SH3RF2 LZTS2 β-catenin M2 polarization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Lung cancer is one of the leading causes of cancer-related morbidity and mortality worldwide. Non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancers ( 1 ). Lung squamous cell carcinoma (LUSC) is a subtype of NSCLC and commonly occurs in people who smoke for a long time. Although the diagnosis and treatment of lung cancer have gradually improved in recent years, the long-term survival of patients with LUSC is still poor ( 2 ). Therefore, in-depth exploration of the molecular mechanisms of LUSC development and progression is of great significance for the prevention and treatment of LUSC. SH3 Domain Containing Ring Finger 2 (SH3RF2) is the member of E3 ubiquitin ligase family and contains three SH3 domain and a RING domain ( 3 ). SH3RF2 is important in neuronal cell survival and brain development ( 4 , 5 ). SH3RF2 plays a cancer-promoting role in colon cancer ( 6 ). Emerging study revealed that SH3RF2 contributed to cisplatin resistance in ovarian cancer cells ( 7 ). However, its function in LUSC has not been reported yet. Leucine zipper tumor suppressor 2 (LZTS2), a member of the leucine zipper tumor suppressor protein family, functions as a tumor suppressor in multiple types of cancers including lung cancer ( 8 – 11 ). In NSCLC, LZTS2 could inhibit cell proliferation and cell cycle transition at the G1/S phase ( 8 ). LZTS2 inhibits the proliferation and migration of lung adenocarcinoma (LUAD) cells ( 12 ). BioGRID database demonstrates that SH3RF2 can bind to LZTS2. However, whether SH3RF2 is involved in the regulation of ubiquitination of LZTS2 is unknown and deserves further exploration. The β-catenin pathway is a common cancer-promoting pathway. LZTS2 can regulate the nuclear import of β-catenin and inhibit β-catenin-mediated transcriptional activation ( 13 ). In addition, there are β-catenin binding sites on the promoters of CCL2 and CCL5, and their expression or secretion might be regulated by β-catenin ( 14 , 15 ). β-Catenin in tumor cells can promote CCL2 secretion, thereby promoting macrophage recruitment and M2 polarization ( 14 ). In summary, this project intends to study the impact of SH3RF2-LZTS2-β-catenin axis on tumor-associated macrophage infiltration and polarization in LUSC. Materials and methods Cell culture and transfection SKMES1 cells were cultured in MEM medium containing 10% fetal bovine serum (FBS). RPMI-1640 medium containing 10% FBS was used to culture NCI-H226 and THP-1 cells. All cell mediums were kept in an incubator with 5% CO 2 at 37℃. SKMES1, NCI-H226, and THP-1 cells were purchased from iCell Bioscience (Shanghai, China). Lipofectamine 3000 was adopted to transfect SH3RF2/LZTS2 overexpression plasmids or SH3RF2-shRNA into the cells. Stably transfected cells were selected using G418. For clone formation, cells were seeded in the culture dish at 300 cells/dish. Visible clones can be formed in about 2 weeks. Colonies were fixed with 4% paraformaldehyde at room temperature for 25 min and stained with crystal violet dye for 5 min. The CCL2 levels were determined using the Human CCL2 ELISA Kit (Lianke Bio, China). CCK-8 The cells were seeded into a 96-well culture plate at a cell number of 5×10 3 per well and cultured in a 37°C, 5% CO2 incubator for 0 h, 24 h, 48 h, and 72 h respectively. Then the cells in each well were incubated with 10 µl CCK-8 for 2 h. The optical density at 450 nm was measured with a microplate reader. In vivo tumor formation Four-week-old male BALB/C nude mice were maintained under a 12-h light/dark cycle at 22 ± 1℃ with humidity 45–55% and allowed free access to water and food. The mice were subcutaneously injected with lung cancer cells (1×10 7 cells per mouse) with stable transfection. Tumor volume was measured every 3 days, and 33 days after inoculation, tumor tissue was collected, weighed and photographed. For radiation therapy, When the tumor volume in the control group reached approximately 150 mm 3 , the nude mice in the radiotherapy group were treated with 15 Gy radiotherapy (X-ray). All animal experiments were approved by the Laboratory Animal Ethics Committee of the Fourth Hospital of Hebei Medical University (No. IACUC-4th Hos Hebmu). Immunofluorescence Cells were fixed with 4% paraformaldehyde for 15 min and incubated with 0.1% TritonX-100 for 30 min. After treatment with 1% BSA for 15 min, cells were incubated with primary antibody overnight at 4℃ and secondary antibody for 60 min at room temperature. The nuclei were counterstained with DAPI. Finally, the cell sections were sealed with anti-fluorescence quencher and observed under a fluorescence microscope. Transwell THP-1 cells were incubated with 150 nM PMA for 24 h and induced to differentiate into M0 macrophages. M0 THP-1 cells were collected by centrifugation and then mixed with serum-free medium to make a single-cell suspension. M0 THP-1 cells (200 µl) were seeded in the upper chamber, and conditioned media (800 µl) from different groups were placed in the lower chamber. After 24 h of culture, the cells were fixed with 4% paraformaldehyde at room temperature for 20 min, and stained with crystal violet for 1 min. Cells were observed under an inverted microscope (IX53, Olympus, Japan). Immunohistochemistry Tumor tissue was dehydrated using graded alcohol, cleared in xylene, and embedded in paraffin. Tissue sections were de-paraffinized with xylene and rehydrated. Low heat-induced antigen retrieval was conducted for 10 min. The sections were incubated with 3% H 2 O 2 for 15 min at room temperature and blocked with 1% BSA for 15 min. Primary antibody incubation was performed at 4℃ overnight and secondary antibody was conducted at 37℃ for 60 min. DAB was used to color development and hematoxylin was employed as counterstain. After dehydration, transparency and sealing, the staining was observed under a microscope. Flow Cytometry M0 THP-1 cells were cultured using conditioned media for 24 h, incubated with CD206 antibody at 4℃ for 30 min and subjected to flow cytometry. For cell cycle detection, cells were incubated with 10 µM BrdU for 30 min at 37℃. After washed with PBS, cells were mixed with pre-cooled 100% ethanol and kept at 4℃ overnight. Cells were collected by centrifugation, followed by incubation with HCl and Triton X-100 for 10 min. Followed by centrifugation, cells were mixed with water and boiled for 10 min. The cooled cells were mixed with 1 ml of 0.5% Triton X-100 and centrifuged. The pellets were resuspended in 50 µl PBS containing 1% BSA and 0.5% Tween and 1 µl of primary FITC-labeled anti-BrdU antibody and kept at 4℃ for 1 h in the dark. After wash with PBS, the cells were incubated with 100 µl RNase A for 30 min at 37℃ and PI for 30 min at 4℃ in the dark. Tumor tissue is cut into pieces and digested with type IV collagenase at 37°C for 1 h. Cells were collected and resuspended with 90 µl buffer. Resuspended cells were incubated with CD11b antibody-coated magnetic beads at 4℃ for 15 min. CD11b + cells were resuspended with PBS and incubated with F4/80 and CD206 antibody for 30 min at 4℃ in the dark, and detected by a flow cytometer. Immunoprecipitation (IP) Non-denaturing lysis buffer (Solarbio, Beijing, China) was used to extract total protein, and the concentration of protein was determined by BCA kit. Antibodies were immobilized on AminoLink Coupling Resin (Pierce, USA). The supernatants were incubated with antibody-immobilized resin overnight. Then the resin was washed with elution buffer. The eluted samples were analyzed by immunoblotting. SH3RF2 antibody (sc-100976, Santa Cruz, China), LZTS2 antibody (15677-1-AP, Proteintech, China), MYC antibody (AE070, ABclonal, China), and Flag antibody (AE063, ABclonal, China) was used in this part. Western blot The polyacrylamide gel consisted of 5% stacking gel and 8% resolving gel. The protein samples were mixed with loading buffer and then heated in a boiling water bath for 5 min. A volume of 20 µl loading samples was subjected to SDS-PAGE. The separated proteins were transferred to a PVDF membrane. Following sealing with blocking solution, the membrane was incubated with primary antibodies overnight at 4℃ and secondary antibodies for 1 h at 37℃. Finally, ECL luminescence solution was used to develop the blot. β-catenin antibody (51067-2-AP, Proteintech, China), rabbit anti–goat IgG-HRP (SE238, Solarbio, China), goat anti-rabbit IgG-HRP (SE134, Solarbio, China), goat anti-mouse IgG-HRP (SE131, Solarbio, China), Histone H3 antibody (GTX122148, Gene Tex, USA) and GAPDH antibody (60004-1-Ig, Proteintech, China) were used here. Other antibodies are listed previously. Real time PCR Total RNA was extracted by chloroform extraction and isopropanol precipitation. NanoDrop One was used to determine the concentration of RNA. RNA is reverse transcribed into cDNA using All-in-One First-Strand SuperMix (Magen Biotechnology, Guangzhou, China). SYBR GREEN were used for real time PCR reaction. The 2 −△△CT method was used to analyze expression data. The forward primers were as follows, SH3RF2: 5’-CGTGGTGGTGGAGATGG-3’, CCL2: 5’-TCATAGCAGCCACCTTCATT-3’, CD163: 5’-GAGACTGTTAGGGAAGGTG-3’, Arg-1: 5’-TTTGCTGACATCCCTAAT-3’, IL-10: 5’-TGAGAACCAAGACCCAGAC-3’. The reverse primers were as follows, SH3RF2: 5’-TGGGAGGTGTAATGTTTGGTG-3’, CCL2: 5’-TCACAGCTTCTTTGGGACAC-3’, CD163: 5’-TGTTTGTTGCCTGGATT-3’, Arg-1: 5’-TTCCGTTCTTCTTGACTT-3’, IL-10: 5’-CATTCTTCACCTGCTCCAC-3’. TOPflash/FOPflash reporter assay TOPflash/FOPflash vector was purchased from YouBio (Shanghai, China). To verify the effect of SH3RF2 on the activation of β-catenin signal pathway, TOP flash (or FOPflash) vector and pRL-TK vector were co-transfected into SH3RF2 stably overexpressed or knocked down LUSC cells using Lipofectamine 3000. A microplate reader was used to detect luciferase activity. The activity of firefly luciferase was normalized to that of renilla luciferase. Statistical analysis All data are presented as mean with SD and analyzed by GraphPad Prism. Data with normal distribution and homogeneity of variance were analyzed by unpaired t test or one- or two-way ANOVA. Tukey’s post hoc test was applied to test multiple comparisons. P < 0.05 was regarded as significant. Results SH3RF2 is upregulated in LUSC patients and is associated with poor survival rate To explore the underlying mechanism of LUSC, we downloaded the gene expression data of lung tissue of LUSC patients from GEO (GSE33532 and GSE19188) and TCGA database. Based on the criteria of p 1, the differentially expressed genes (DEGs) in the three datasets were presented in Fig. 1 A. There are 848 up-regulated DEGs and 1189 down-regulated DEGs shared in these three datasets (Fig. 1 B). GO and KEGG analysis were performed on these DEGs. Top 20 GO terms and top 15 KEGG pathways according to the p-value were respectively presented in Fig. 1 C and Fig. 1 D. Among the shared DEGs, SH3RF2, a gene that interests us, was screened out. The expression analysis of SH3RF2 in GSE33532, GSE19188, TCGA and GEPIA showed that SH3RF2 was specifically up-regulated in LUSC patients. However, it did not show an up-regulation trend in LUAD patients (Fig. 1 E). Kaplan Meier plot showed that SH3RF2 has a stronger association with poor survival in LUSC patients compared with LUAD patients (Fig. 1 F). Therefore, we selected SH3RF2 for the further study. SH3RF2 promotes the proliferation of LUSC cells SH3RF2 knockdown and overexpression were conducted in NCI-H226 and SKMES1 cells. SH3RF2 knockdown decreased the colony formation rate and its overexpression increased the colony formation rate (Supplementary Fig. 1A). The cell viability was inhibited in the SH3RF2 knockdown group and promoted in the SH3RF2 overexpression group (Supplementary Fig. 1B). Compared with the NC sh group, the proportion of G1 phase cells increased and the proportion of S and G2 phases decreased in SH3RF2 knockdown group. The overexpression group showed the opposite trend (Supplementary Fig. 1C). All above demonstrated the positive effect of SH3RF2 on the proliferation of LUSC cells. SH3RF2 promotes CCL2 secretion and M2 polarization The RNA level of CCL2 was decreased after SH3RF2 downregulation and increased after SH3RF2 upregulation (Fig. 2 A). We also evaluated the level of CCL2 in the cell supernatant and found that upregulation of SH3RF2 increased the protein level of CCL2 (Fig. 2 B). To explore the effect of SH3RF2 on M2 polarization, human THP-1 cells were treated with 150 nM PMA to induce the differentiation into M0 macrophages. The conditioned medium (CM) from NCI-H226 and SKMES1 cells were used to culture the M0 cells for 24 h. Transwell assay showed that SH3RF2 played a promoting role in the migration and invasion ability of M0 cells (Fig. 2 C). The RNA levels of Arg-1, CD163 and IL-10 were decreased in M0 cells of the SH3RF2 knockdown group and increased in M0 cells of the SH3RF2 overexpression group (Fig. 2 D). After culture using CM, the proportion of CD206 + cells contained in M0 cells in the knockdown group decreased, while that in the overexpression group increased (Fig. 2 E). SH3RF2 regulates β-catenin in the LUSC cells Upregulation and downregulation of SH3RF2 did not affect the total protein level of β-catenin in LUSC cells, but affects its protein level in the nucleus (Supplementary Fig. 2A). Immunofluorescence staining of β-catenin (red) showed that SH3RF2 overexpression increased the red fluorescence intensity in the nucleus (Supplementary Fig. 2B). The TOPflash/FOPflash luciferase reporter system was used to measure the activation of β-catenin signal pathway. FOPflash, a mutant TOPflash was used as a control. Cells with SH3RF2 knockdown showed a reduced luciferase activity, and cells with SH3RF2 overexpression showed an elevated luciferase activity (Supplementary Fig. 2C). SH3RF2 promoted the activation of β-catenin signal pathway. SH3RF2 affects the development of tumor cells and macrophages through β-catenin ICG-001, the inhibitor of β-catenin/TCF mediated transcription, was used to treat SKMES1 cells for 24 h. SH3RF2 overexpression improved the cell viability, while ICG-001 reversed this promoting effect (Fig. 3 A). After treatment of ICG-001, the cells were cultured in normal medium for 24 h. The levels of CCL2 in cells and cell supernatant were determined by real time PCR and ELISA respectively. It found that the expression of CCL2 was promoted by SH3RF2 and inhibited by ICG-001 (Fig. 3 B and C). The conditioned medium of the above cells was used to treat M0 cells. ICG- 001 alleviated the positive effect of SH3RF2 on cell migration and invasion (Fig. 3 D). The RNA levels of Arg-1, CD163 and IL-10 in M0 cells were increased in SH3RF2 overexpression group, and decreased after ICG-001 treatment (Fig. 3 E). In addition, the proportion of CD206 + cells showed a same trend in the two groups (Fig. 3 F). It demonstrated that ICG-001 inhibit the process regulated by SH3RF2. Effect of SH3RF2 on the growth of LUSC tumor SKMES1 cells with SH3RF2 silence and overexpression were injected into nude mice. The representative pictures of tumor were presented in Fig. 4 A and B. Tumor volume and weight were decreased in the SH3RF2 knockdown group and increased in the SH3RF2 overexpression group. The levels of CCL2 in tumor tissues was elevated by SH3RF2 upregulation and reduced by SH3RF2 downregulation (Fig. 4 C and D). Immunohistochemical staining analyzed the expression of SH3RF2 (Fig. 4 E) and Ki67 in tumor tissues. The positive staining of Ki67 was enhanced in the SH3RF2 overexpression group (Fig. 4 F). Flow cytometry found that the proportion of CD11b + CD206 + F4/80 + cells was decreased after SH3RF2 downregulation and increased after SH3RF2 upregulation (Fig. 4 G). Target protein of SH3RF2 We obtained a PPI network of SH3RF2 with its potential target proteins from the BioGRID database (Supplementary Fig. 3A). Among these candidate proteins, we noticed the protein LZTS2, which was reported to regulated the β-catenin. Therefore, co-IP was performed in SKMES1 and NCI-H226 cells and found that SH3RF2 interacted with LZTS2 (Supplementary Fig. 3B). SH3RF2 regulates the ubiquitination of LZTS2 The protein level of LZTS2 was increased in cells with SH3RF2 knockdown and decreased in cells with SH3RF2 overexpression (Fig. 5 A and B). After the LUSC cells were treated with 20 µg/ml cycloheximide (CHX) for 0, 30, 60, 120 and 240 min, the protein levels of LZTS2 in the SH3RF2 overexpression group gradually decreased with increasing CHX treatment time (Fig. 5 C and D). SKMES1 cells with SH3RF2 overexpression were transfected with MYC-LZTS2 vector and HA-Ub vector. After 42 h, the cells were treated with 10 µM MG132 for 6 h. Compared with the EV group, the ubiquitination of LZTS2 in the SH3RF2 overexpression group was enhanced (Fig. 5 E). To confirm whether the RING domain of SH3RF2 is involved in the regulation of LZTS2 ubiquitination, we mutated the C28S, C33S and C36S sites of SH3RF2 (RING domain inactivation) ( 3 , 16 ). Co-IP found that ubiquitination levels of LZTS2 were reduced in the SH3RF2 mutant group compared to wild-type SH3RF2 group (Fig. 5 F). SH3RF2 regulates LUSC cell growth, macrophage chemotaxis and M2 polarization through LZTS2 LZTS2 was overexpressed in the SKMES1 cells with SH3RF2 overexpression (Fig. 6 A). The cell viability of cells with SH3RF2 and LZTS2 overexpression were decreased compared with the cells with SH3RF2 overexpression (Fig. 6 B). Immunofluorescence for β-catenin showed that SH3RF2 promoted the nuclear translocation of β-catenin, while overexpression of LZTS2 alleviated this promotion effect (Fig. 6 C). The protein level of CCL2 and the RNA levels of Arg-1, CD163 and IL-10 were decreased in the cells with SH3RF2 and LZTS2 overexpression compared with the cells with SH3RF2 overexpression (Fig. 6 D and E). Transwell assay found that the number of migrated M0 cells in the LZTS2 upregulation group reduced (Fig. 6 F). In the same, the proportion of CD206 + cells decreased in the LZTS2 upregulation group (Fig. 6 G). The impact of SH3RF2 inhibition and radiation therapy in the growth of LUSC. In the group with SH3RF2 knockdown and radiotherapy treatment, the tumor volume and weight were smaller than those in the group with SH3RF2 knockdown alone (Supplementary Fig. 4A). In addition, the immunohistochemistry staining for Ki-67 revealed that the positive staining was inhibited in the cells with SH3RF2 knockdown and radiotherapy treatment (Supplementary Fig. 4B). Discussion This study clarified the promoting effect of SH3RF2 on LUSC proliferation and growth, expression of chemokine CCL2, and M2-type polarization. We also revealed the SH3RF2-targeted protein LZTS2 and the ubiquitination of LZTS2 through the RING domain of SH3RF2. Collectively, our results clarified the relationship between SH3RF2-LZTS2-β-catenin axis in LUSC. Unfortunately, this study was unable to collect LUSC patient samples to verify the correlation between SH3RF expression and clinically relevant indicators. Currently, there are few studies on SH3RF2. In several cancer-related reports, it has been shown to promote tumor progression. Our research results also confirmed its cancer-promoting effect in LUSC. SH3RF2, as an E3 ubiquitin ligase, has been shown to regulate the ubiquitination of related proteins. SH3RF2 can degrade POSH protein through ubiquitination and the RING domain of SH3RF2 and the RING domain of POSH are involved in this degradation process ( 16 ). SH3RF2 directly binds ACLY and promotes its K48-linked ubiquitination-dependent degradation ( 17 ). SH3RF2 promotes K48-linked ubiquitination of RBPMS, thereby increasing its proteasomal degradation ( 7 ). In this study, we found that SH3RF2 regulated the ubiquitination of LZTS2 through its RING domain. However, these studies including the current study only revealed that SH3RF2 is involved in ubiquitination, but did not clarify the specific way or binding region of SH3RF2 binding to target proteins. A report demonstrated that the third SH3 domain (370–459 amino acids) of SH3RF2 protein is a specific PAK4 binding region and inhibits PAK4 ubiquitination, resulting in the upregulation of PAK4 protein ( 6 ). Although this report revealed the specific domain of SH3RF2 that binds target proteins, the difference is that SH3RF2 inhibits the ubiquitination of target proteins through this domain, which is significantly different from those studies mentioned above. All of these results reflect that the three SH3 domains and the RING domain contained in SH3RF2 may have special functions and significance. As far as the RING domain alone is concerned, it is involved in the ubiquitination process. The SH3 domain is reported to bind to its target partner with a typical PXXP motif (X represents any amino acid), and has functions such as regulating endocytosis and actin cytoskeleton remodeling ( 18 ). In the SH3RF2 protein, what functions the combination of these domains can play is worthy of further exploration. It is well known that macrophage M1/M2 polarization is important to the development of cancer. M2 macrophage is also known as tumor-associated macrophage, which can promote tumor growth, invasion, metastasis and angiogenesis, and inhibit tumor immune response ( 19 ). In non-small cell lung cancer, M2 macrophages have been shown to promote tumor growth and migration ( 20 , 21 ). M2 macrophages mainly promote tumor occurrence and metastasis by secreting angiogenic factors, chemokines, and downregulating major histocompatibility complex class II (MHC II) and costimulatory ligands CD80 and CD86 ( 22 ). Here, we found that SH3RF2 could promote M2 polarization and increase the proportion of M2 macrophages. In addition, our results found that SH3RF2 inhibited the expression of CCL2 in tumor cells. CCL2 is known to promote M2 polarization. It indirectly reflects the promotion effect of SH3RF2 on M2 polarization. It is worth mentioning that reducing the proportion of M2 macrophages is gradually recognized as a new strategy for tumor immunotherapy ( 23 ). Therefore, our results provide new candidate targets for immunotherapy of LUSC. The Wnt/β-catenin pathway is increasingly recognized as a potentially important target for anticancer therapy ( 24 ). Nuclear translocated β-catenin activates genes related to cell proliferation, survival, differentiation, and migration through TCF/LEF transcription factors ( 25 ). In various tumors such as colorectal cancer ( 26 ), prostate cancer ( 27 ), and lung cancer ( 8 , 12 ), LZTS2 has been reported to be involved in the activation of the Wnt/β-catenin pathway. We found that SH3RF2 promoted the nuclear import of β-catenin, and ICG-001 alleviated the promoting effects of SH3RF2 overexpression on tumor cell proliferation, macrophage chemotaxis and M2 polarization. In addition, SH3RF2 regulated the ubiquitination of LZTS2. Rescue experiment demonstrated that SH3RF2 regulates LUSC cell growth, macrophage chemotaxis and M2 polarization through LZTS2. Obviously, these effects can be achieved through the SH3RF2-LZTS2-β-catenin cascade reaction. Compared with LUAD patients, patients with advanced LUSC had higher ORR, shorter PFS and OS rates in first-line immunotherapy ( 28 ). Since LUSC has very few driver gene mutations and only 3% of patients have epidermal growth factor receptor mutations and anaplastic lymphoma kinase rearrangements, radiotherapy is more important for the treatment of LUSC than LUAD ( 2 ). Most patients with early-stage LUSC can be effectively treated with radiation therapy, but patients with advanced LUSC may have limited radiotherapy treatment ( 29 ). Therefore, in order to improve the survival rate of LUSC, developing new treatment strategies is a top priority. We tested the effect of combined treatment with radiotherapy and SH3RF2 overexpression on xenografted tumors and found that it could significantly inhibit tumor growth. This may provide new directions for developing new treatment strategies. Declarations Ethical Approval All animal experiments were approved by the Laboratory Animal Ethics Committee of the Fourth Hospital of Hebei Medical University (No. IACUC-4th Hos Hebmu) and performed in compliance with ARRIVE guidelines. Conflict of Interest None Funding This research was funded by the Natural Science Foundation of Hebei Province (No. H2023206301). Author Contribution Jie Yang: Funding acquisition, investigation and methodology. Zhong-Fei Jia: Investigation and data curation. Juan Li: Investigation. Chao Jiang: Formal analysis. Xin Zhao, Yu-Xiang Wang and Xiao-Guo Ma: visualization; writing–original draft. Xin-Jian Xu: Conceptualization and project administration. All authors reviewed and edited the manuscript. Acknowledgement All authors thank the Fourth Hospital of Hebei Medical University and the Natural Science Foundation of Hebei Province for their assistance. 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Oct4 promotes M2 macrophage polarization through upregulation of macrophage colony-stimulating factor in lung cancer. J Hematol Oncol. 2020;13(1):62. Madeddu C, Donisi C, Liscia N, Lai E, Scartozzi M, Maccio A. EGFR-Mutated Non-Small Cell Lung Cancer and Resistance to Immunotherapy: Role of the Tumor Microenvironment. Int J Mol Sci. 2022;23(12). Zhang S, Xie F, Li K, Zhang H, Yin Y, Yu Y, et al. Gold nanoparticle-directed autophagy intervention for antitumor immunotherapy via inhibiting tumor-associated macrophage M2 polarization. Acta Pharm Sin B. 2022;12(7):3124–38. Pai SG, Carneiro BA, Mota JM, Costa R, Leite CA, Barroso-Sousa R, et al. Wnt/beta-catenin pathway: modulating anticancer immune response. J Hematol Oncol. 2017;10(1):101. Liu J, Xiao Q, Xiao J, Niu C, Li Y, Zhang X, et al. Wnt/beta-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct Target Ther. 2022;7(1):3. Dong Z, Li J, Dai W, Yu D, Zhao Y, Liu S, et al. RRP15 deficiency induces ribosome stress to inhibit colorectal cancer proliferation and metastasis via LZTS2-mediated beta-catenin suppression. Cell Death Dis. 2023;14(2):89. Yu EJ, Hooker E, Johnson DT, Kwak MK, Zou K, Luong R, et al. LZTS2 and PTEN collaboratively regulate ss-catenin in prostatic tumorigenesis. PLoS ONE. 2017;12(3):e0174357. Qin J, Yi S, Zhou H, Zeng C, Zou M, Zeng X, et al. Efficacy of radiotherapy in combination with first-line immunotherapy and chemotherapy for advanced lung squamous cell carcinoma: a propensity score analysis. Front Immunol. 2023;14:1138025. Santivasi WL, Xia F. Ionizing radiation-induced DNA damage, response, and repair. Antioxid Redox Signal. 2014;21(2):251–9. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.tif Supplementary Figure 1 (A) The colony formation rate of cells with SH3RF2 overexpression or knockdown. (B) Cell viability of lung cancer cells detected by CCK-8 assay. (C) Cell cycle distribution detected by flow cytometry. NC sh : negative control shRNA, SR sh-1 : SH3RF2 shRNA-1, SR sh-2 : SH3RF2 shRNA-2, EV: empty vector, SR oe : SH3RF3 overexpression. **: p<0.01, ***: p<0.001. SupplementaryFigure2.tif Supplementary Figure 2 (A) Total and nuclear expression of β-catenin in NCI-H226 and SKMES1 cells analyzed by western blot. (B) Immunofluorescence detection of the distribution of β-catenin in NCI-H226 and SKMES1 cells. Bar= 100 μm. (C) Relative luciferase activity, TOP: TOP flash vector, FOP: FOP flash vector. NC sh : negative control shRNA, SR sh-1 : SH3RF2 shRNA-1, SR sh-2 : SH3RF2 shRNA-2, EV: empty vector, SR oe : SH3RF3 overexpression. *: p<0.05, **: p<0.01, compared to NCsh-TOP group. ### p<0.001, compared to EV-TOP group. SupplementaryFigure3.tif Supplementary Figure 3 (A) The proteins that may interact with SH3RF2 were predicted by the BioGRID database. (B) Interaction between SH3EF2 and LZTS2 in NCI-H226 and SKMES1 cells verified by co-immunoprecipitation (co-IP). SupplementaryFigure4.tif Supplementary Figure 4 (A) SKMES1 cells with SH3RF2 knockdown were injected subcutaneously into the right armpit of Balb/c nude mice. When the tumor in the NC sh group grown to approximately 150 mm 3 , the nude mice in the radiation therapy (RT) group were treated with 15 Gy X rays. Tumor size was measured every 3 days. Tumors were removed and weighed after 33 days. (B) Immunohistochemistry for Ki-67 in tumor tissues, bar=50 μm. NC sh : negative control shRNA, SR sh : SH3RF2 shRNA. *: p<0.05, ***: p<0.001. Cite Share Download PDF Status: Published Journal Publication published 17 Jul, 2025 Read the published version in Biology Direct → Version 1 posted Editorial decision: Revision requested 24 Mar, 2025 Reviews received at journal 24 Mar, 2025 Reviewers agreed at journal 10 Mar, 2025 Reviewers agreed at journal 08 Feb, 2025 Reviews received at journal 06 Jan, 2025 Reviewers agreed at journal 17 Dec, 2024 Reviewers invited by journal 05 Dec, 2024 Editor assigned by journal 26 Nov, 2024 Submission checks completed at journal 19 Nov, 2024 First submitted to journal 14 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-5457209","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":382740648,"identity":"63b4a210-97b5-4b75-803c-059def78e9a3","order_by":0,"name":"Jie Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYDCCAwhm4mMYV4KAFsYGKDPZmGQtbNJEaeE73vz8wcc9h+XN+Rc8qy6ouBNtcID54G0eBrs8XFokzxwzbJzx7LDhzhkP0m7POPMsd8MBtmRrHobkYlxaDG7kMDbzHDjMuOHGgbTbvG2HgVp4zKR5GA4kNhDQYg/SUsz7D6SF/xtRWhI3nG9IY+ZtANvChlcLyC8zZxxIT95wgyFZmufYs9yZh9mMLecYJOPUAgyxBx8+HLC23XD+TOJnnpo7uX3Hmx/eeFNhh1MLFDQD4yInAcJmBjsYv3ogqGNg4D9+gKCyUTAKRsEoGJkAADAEaTXKNHGEAAAAAElFTkSuQmCC","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jie","middleName":"","lastName":"Yang","suffix":""},{"id":382740649,"identity":"1828c564-6f0d-4bb2-b925-287ed9fb2323","order_by":1,"name":"Zhongfei Jia","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhongfei","middleName":"","lastName":"Jia","suffix":""},{"id":382740650,"identity":"3edcddeb-b181-417a-aefd-d8e337029458","order_by":2,"name":"Juan Li","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Li","suffix":""},{"id":382740651,"identity":"cc9e4f51-b81a-41c6-b53e-7e1de21c8cec","order_by":3,"name":"Chao Jiang","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Jiang","suffix":""},{"id":382740652,"identity":"9c2ae6ae-0756-44c7-8874-34da4d716b38","order_by":4,"name":"Xin Zhao","email":"","orcid":"","institution":"Clinical College of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhao","suffix":""},{"id":382740653,"identity":"96aeed05-b2c8-4c49-9846-fad4519c1141","order_by":5,"name":"Yuxiang Wang","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuxiang","middleName":"","lastName":"Wang","suffix":""},{"id":382740654,"identity":"a74e48df-6f33-43d1-9c3f-ff7ac9dc25a7","order_by":6,"name":"Xiaoguo Ma","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoguo","middleName":"","lastName":"Ma","suffix":""},{"id":382740655,"identity":"a3086fe9-857f-4ddf-befb-c1aeacd2d4c8","order_by":7,"name":"Xinjian Xu","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinjian","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2024-11-15 03:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5457209/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5457209/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13062-025-00677-0","type":"published","date":"2025-07-17T16:05:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70488184,"identity":"daa2b69d-e905-424e-a6e1-4d9f3946f2e2","added_by":"auto","created_at":"2024-12-03 16:19:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5613723,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Volcano plot of differential genes (p\u0026lt;0.01 and log2FC\u0026lt;-1 or \u0026gt;1) in GSE33532, GSE19188, and TCGA-LUSC datasets. (B) Venn diagram of genes in the three datasets. (C) Ridge plots of the top 20 terms of GO analysis according to p-value. (D) System diagram of top 15 KEGG pathways. (E) The expression level of SH3RF2 in GSE33532, GSE19188, and TCGA-LUSC datasets (left panel) and GEPIA database (right panel). (F) Relationship between SH3RF2 expression and survival of lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) patients in Kaplan-Meier plotter database.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5457209/v1/8de1b1acd6c1fcd0ec353886.png"},{"id":70487908,"identity":"0b6bafde-2c5c-4a85-861f-82ef2a745f3f","added_by":"auto","created_at":"2024-12-03 16:11:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5197063,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Relative RNA level of CCL2 in NCI-H226 and SKMES1 cells. (B) Protein level of CCL2 determined by ELISA. (C) Migrated cells detected by transwell assay. (D) RNA level of Arg-1, CD163 and IL-10 in M0 THP-1 cells treated with NCI-H226 conditioned medium (CM) or SKMES1 CM. (E) The proportion of CD206+ cells in M0 THP-1 cells with CM treatment. NC\u003csup\u003esh\u003c/sup\u003e: negative control shRNA, SR\u003csup\u003esh-1\u003c/sup\u003e: SH3RF2 shRNA-1, SR\u003csup\u003esh-2\u003c/sup\u003e: SH3RF2 shRNA-2, EV: empty vector, SR\u003csup\u003eoe\u003c/sup\u003e: SH3RF3 overexpression. **: p\u0026lt;0.01, ***: p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5457209/v1/bf6845e418ae55555f6e74ea.png"},{"id":70489012,"identity":"381849d9-14b6-4f0b-b29c-34cd6e40e636","added_by":"auto","created_at":"2024-12-03 16:27:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3865034,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Cell viability of SKMES1 cells treated with ICG-001. (B) RNA level of CCL2 in SKMES1 cells. (C) Protein level of CCL2 in SKMES1 cells. (D) Cellular migration and invasion ability of THP-1 cells analyzed by transwell assay. (E) RNA level of Arg-1, CD163 and IL-10 in M0 THP-1 cells treated with SKMES1 CM. (F) The proportion of CD206+ cells in M0 THP-1 cells with SKMES1 CM treatment. EV: empty vector, SR\u003csup\u003eoe\u003c/sup\u003e: SH3RF3 overexpression. **: p\u0026lt;0.01, ***: p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5457209/v1/51cb1c31b104fb089b3cbfd6.png"},{"id":70488187,"identity":"d93bb646-4413-46eb-9114-4b847c2dcca3","added_by":"auto","created_at":"2024-12-03 16:19:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11625846,"visible":true,"origin":"","legend":"\u003cp\u003e(A \u0026amp; B) SKMES1 cells with SH3RF2 overexpression or knockdown were injected subcutaneously into the right armpit of Balb/c nude mice. Tumor size was measured every 3 days. Tumors were removed and weighed after 33 days. (C \u0026amp; D) The protein level of CCL2 in tumor determined by ELISA. (E) Detection of SH3RF2 expression in tumor tissues by immunohistochemistry, bar=50 μm. (F) Immunohistochemistry for Ki-67 in tumor tissues, bar=50 μm. (G) The proportion of CD11b+F4/80+CD206+ cells in tumor. NC\u003csup\u003esh\u003c/sup\u003e: negative control shRNA, SR\u003csup\u003esh-1\u003c/sup\u003e: SH3RF2 shRNA-1, SR\u003csup\u003esh-2\u003c/sup\u003e: SH3RF2 shRNA-2, EV: empty vector, SR\u003csup\u003eoe\u003c/sup\u003e: SH3RF3 overexpression. **: p\u0026lt;0.01, ***: p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5457209/v1/9703df9e485d73965ec817db.png"},{"id":70487910,"identity":"85f08aab-ae5d-4501-b887-ea8056285c5d","added_by":"auto","created_at":"2024-12-03 16:11:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3938663,"visible":true,"origin":"","legend":"\u003cp\u003e(A \u0026amp; B) Protein level of LZTS2 in NCI-H226 and SKMES1 cells. (C \u0026amp; D) Protein level of LZTS2 in lung cancer cells with CHX treatment. (E) The ubiquitination of LZTS2 in SKMES1 cells was detected by Co-IP. (F) Regulation of LZTS2 ubiquitination by the RING domain of SH3RF2 was verified by co-IP, SR\u003csup\u003ewt\u003c/sup\u003e: wild type SH3RF2, SR\u003csup\u003emut\u003c/sup\u003e: RING domain-mutated SH3RF2. NC\u003csup\u003esh\u003c/sup\u003e: negative control shRNA, SR\u003csup\u003esh-1\u003c/sup\u003e: SH3RF2 shRNA-1, SR\u003csup\u003esh-2\u003c/sup\u003e: SH3RF2 shRNA-2, EV: empty vector, SR\u003csup\u003eoe\u003c/sup\u003e: SH3RF3 overexpression. **: p\u0026lt;0.01, ***: p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5457209/v1/3fb51a6c7854492d59927e78.png"},{"id":70487914,"identity":"0c823c16-a190-47db-b98e-92b72922e308","added_by":"auto","created_at":"2024-12-03 16:11:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6831575,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Protein level of LZTS2 in SKMES1 cells with SH3RF2 and LZTS2 overexpression. (B) Cell viability of SKMES1 cells with SH3RF2 and LZTS2 overexpression. (C) Immunofluorescence detection of the distribution of β-catenin in SKMES1 cells. (D) Protein level of CCL2 in SKMES1 cells. (E) RNA levels of Arg-1, CD163 and IL-10 in M0 THP-1 cells with SKMES1 CM treatment. (F) Cellular migration and invasion ability of M0 THP-1 cells with SKMES1 CM treatment. (G) The proportion of CD206+ cells in the M0 THP-1 cells treated with SKMES1 CM. EV: empty vector, SR\u003csup\u003eoe\u003c/sup\u003e: SH3RF3 overexpression. LZ\u003csup\u003eoe\u003c/sup\u003e: LZTS2 overexpression. **: p\u0026lt;0.01, ***: p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5457209/v1/741068eba83105f1858e4b66.png"},{"id":88506061,"identity":"6ef6372e-0367-4fd9-974d-0a44456bbb86","added_by":"auto","created_at":"2025-08-07 07:30:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":35302304,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5457209/v1/3be0b390-7838-44a9-8abc-4b8d1dce2fb9.pdf"},{"id":70487911,"identity":"5a45b270-556c-4964-a036-74cdec00f82f","added_by":"auto","created_at":"2024-12-03 16:11:53","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1422504,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 1\u003c/p\u003e\n\u003cp\u003e(A) The colony formation rate of cells with SH3RF2 overexpression or knockdown. (B) Cell viability of lung cancer cells detected by CCK-8 assay. (C) Cell cycle distribution detected by flow cytometry. NC\u003csup\u003esh\u003c/sup\u003e: negative control shRNA, SR\u003csup\u003esh-1\u003c/sup\u003e: SH3RF2 shRNA-1, SR\u003csup\u003esh-2\u003c/sup\u003e: SH3RF2 shRNA-2, EV: empty vector, SR\u003csup\u003eoe\u003c/sup\u003e: SH3RF3 overexpression. **: p\u0026lt;0.01, ***: p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5457209/v1/a262f167f99e307a44bbf847.tif"},{"id":70489310,"identity":"f93a5c8b-872f-4525-bfc1-2b249b313574","added_by":"auto","created_at":"2024-12-03 16:35:53","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1748462,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 2\u003c/p\u003e\n\u003cp\u003e(A) Total and nuclear expression of β-catenin in NCI-H226 and SKMES1 cells analyzed by western blot. (B) Immunofluorescence detection of the distribution of β-catenin in NCI-H226 and SKMES1 cells. Bar= 100 μm. (C) Relative luciferase activity, TOP: TOP flash vector, FOP: FOP flash vector. NC\u003csup\u003esh\u003c/sup\u003e: negative control shRNA, SR\u003csup\u003esh-1\u003c/sup\u003e: SH3RF2 shRNA-1, SR\u003csup\u003esh-2\u003c/sup\u003e: SH3RF2 shRNA-2, EV: empty vector, SR\u003csup\u003eoe\u003c/sup\u003e: SH3RF3 overexpression. *: p\u0026lt;0.05, **: p\u0026lt;0.01, compared to NCsh-TOP group. ### p\u0026lt;0.001, compared to EV-TOP group.\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-5457209/v1/5e9d3583334f0bbd291f7f51.tif"},{"id":70488185,"identity":"e59afcc6-42df-4a5a-99e9-d35bc4be0020","added_by":"auto","created_at":"2024-12-03 16:19:53","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":837576,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 3\u003c/p\u003e\n\u003cp\u003e(A) The proteins that may interact with SH3RF2 were predicted by the BioGRID database. (B) Interaction between SH3EF2 and LZTS2 in NCI-H226 and SKMES1 cells verified by co-immunoprecipitation (co-IP).\u003c/p\u003e","description":"","filename":"SupplementaryFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-5457209/v1/97e054dd786693abd57176f3.tif"},{"id":70487918,"identity":"dedcea61-73f3-4b37-8c93-194ea678d2eb","added_by":"auto","created_at":"2024-12-03 16:11:53","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1683010,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 4\u003c/p\u003e\n\u003cp\u003e(A) SKMES1 cells with SH3RF2 knockdown were injected subcutaneously into the right armpit of Balb/c nude mice. When the tumor in the NC\u003csup\u003esh\u003c/sup\u003e group grown to approximately 150 mm\u003csup\u003e3\u003c/sup\u003e, the nude mice in the radiation therapy (RT) group were treated with 15 Gy X rays. Tumor size was measured every 3 days. Tumors were removed and weighed after 33 days. (B) Immunohistochemistry for Ki-67 in tumor tissues, bar=50 μm. NC\u003csup\u003esh\u003c/sup\u003e: negative control shRNA, SR\u003csup\u003esh\u003c/sup\u003e: SH3RF2 shRNA. *: p\u0026lt;0.05, ***: p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"SupplementaryFigure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-5457209/v1/f0bedd59e0b1762445e28782.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"The role of SH3RF2 in lung squamous cell carcinoma and M2 polarization: insights into LZTS2 ubiquitination","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer is one of the leading causes of cancer-related morbidity and mortality worldwide. Non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancers (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Lung squamous cell carcinoma (LUSC) is a subtype of NSCLC and commonly occurs in people who smoke for a long time. Although the diagnosis and treatment of lung cancer have gradually improved in recent years, the long-term survival of patients with LUSC is still poor (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Therefore, in-depth exploration of the molecular mechanisms of LUSC development and progression is of great significance for the prevention and treatment of LUSC.\u003c/p\u003e \u003cp\u003eSH3 Domain Containing Ring Finger 2 (SH3RF2) is the member of E3 ubiquitin ligase family and contains three SH3 domain and a RING domain (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). SH3RF2 is important in neuronal cell survival and brain development (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). SH3RF2 plays a cancer-promoting role in colon cancer (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Emerging study revealed that SH3RF2 contributed to cisplatin resistance in ovarian cancer cells (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). However, its function in LUSC has not been reported yet.\u003c/p\u003e \u003cp\u003eLeucine zipper tumor suppressor 2 (LZTS2), a member of the leucine zipper tumor suppressor protein family, functions as a tumor suppressor in multiple types of cancers including lung cancer (\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). In NSCLC, LZTS2 could inhibit cell proliferation and cell cycle transition at the G1/S phase (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). LZTS2 inhibits the proliferation and migration of lung adenocarcinoma (LUAD) cells (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). BioGRID database demonstrates that SH3RF2 can bind to LZTS2. However, whether SH3RF2 is involved in the regulation of ubiquitination of LZTS2 is unknown and deserves further exploration.\u003c/p\u003e \u003cp\u003eThe β-catenin pathway is a common cancer-promoting pathway. LZTS2 can regulate the nuclear import of β-catenin and inhibit β-catenin-mediated transcriptional activation (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In addition, there are β-catenin binding sites on the promoters of CCL2 and CCL5, and their expression or secretion might be regulated by β-catenin (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). β-Catenin in tumor cells can promote CCL2 secretion, thereby promoting macrophage recruitment and M2 polarization (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In summary, this project intends to study the impact of SH3RF2-LZTS2-β-catenin axis on tumor-associated macrophage infiltration and polarization in LUSC.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfection\u003c/h2\u003e \u003cp\u003eSKMES1 cells were cultured in MEM medium containing 10% fetal bovine serum (FBS). RPMI-1640 medium containing 10% FBS was used to culture NCI-H226 and THP-1 cells. All cell mediums were kept in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃. SKMES1, NCI-H226, and THP-1 cells were purchased from iCell Bioscience (Shanghai, China). Lipofectamine 3000 was adopted to transfect SH3RF2/LZTS2 overexpression plasmids or SH3RF2-shRNA into the cells. Stably transfected cells were selected using G418. For clone formation, cells were seeded in the culture dish at 300 cells/dish. Visible clones can be formed in about 2 weeks. Colonies were fixed with 4% paraformaldehyde at room temperature for 25 min and stained with crystal violet dye for 5 min. The CCL2 levels were determined using the Human CCL2 ELISA Kit (Lianke Bio, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCCK-8\u003c/h3\u003e\n\u003cp\u003eThe cells were seeded into a 96-well culture plate at a cell number of 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e per well and cultured in a 37\u0026deg;C, 5% CO2 incubator for 0 h, 24 h, 48 h, and 72 h respectively. Then the cells in each well were incubated with 10 \u0026micro;l CCK-8 for 2 h. The optical density at 450 nm was measured with a microplate reader.\u003c/p\u003e\n\u003ch3\u003eIn vivo tumor formation\u003c/h3\u003e\n\u003cp\u003eFour-week-old male BALB/C nude mice were maintained under a 12-h light/dark cycle at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃ with humidity 45\u0026ndash;55% and allowed free access to water and food. The mice were subcutaneously injected with lung cancer cells (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells per mouse) with stable transfection. Tumor volume was measured every 3 days, and 33 days after inoculation, tumor tissue was collected, weighed and photographed. For radiation therapy, When the tumor volume in the control group reached approximately 150 mm\u003csup\u003e3\u003c/sup\u003e, the nude mice in the radiotherapy group were treated with 15 Gy radiotherapy (X-ray). All animal experiments were approved by the Laboratory Animal Ethics Committee of the Fourth Hospital of Hebei Medical University (No. IACUC-4th Hos Hebmu).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eCells were fixed with 4% paraformaldehyde for 15 min and incubated with 0.1% TritonX-100 for 30 min. After treatment with 1% BSA for 15 min, cells were incubated with primary antibody overnight at 4℃ and secondary antibody for 60 min at room temperature. The nuclei were counterstained with DAPI. Finally, the cell sections were sealed with anti-fluorescence quencher and observed under a fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eTranswell\u003c/h3\u003e\n\u003cp\u003eTHP-1 cells were incubated with 150 nM PMA for 24 h and induced to differentiate into M0 macrophages. M0 THP-1 cells were collected by centrifugation and then mixed with serum-free medium to make a single-cell suspension. M0 THP-1 cells (200 \u0026micro;l) were seeded in the upper chamber, and conditioned media (800 \u0026micro;l) from different groups were placed in the lower chamber. After 24 h of culture, the cells were fixed with 4% paraformaldehyde at room temperature for 20 min, and stained with crystal violet for 1 min. Cells were observed under an inverted microscope (IX53, Olympus, Japan).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eTumor tissue was dehydrated using graded alcohol, cleared in xylene, and embedded in paraffin. Tissue sections were de-paraffinized with xylene and rehydrated. Low heat-induced antigen retrieval was conducted for 10 min. The sections were incubated with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 15 min at room temperature and blocked with 1% BSA for 15 min. Primary antibody incubation was performed at 4℃ overnight and secondary antibody was conducted at 37℃ for 60 min. DAB was used to color development and hematoxylin was employed as counterstain. After dehydration, transparency and sealing, the staining was observed under a microscope.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFlow Cytometry\u003c/h3\u003e\n\u003cp\u003eM0 THP-1 cells were cultured using conditioned media for 24 h, incubated with CD206 antibody at 4℃ for 30 min and subjected to flow cytometry. For cell cycle detection, cells were incubated with 10 \u0026micro;M BrdU for 30 min at 37℃. After washed with PBS, cells were mixed with pre-cooled 100% ethanol and kept at 4℃ overnight. Cells were collected by centrifugation, followed by incubation with HCl and Triton X-100 for 10 min. Followed by centrifugation, cells were mixed with water and boiled for 10 min. The cooled cells were mixed with 1 ml of 0.5% Triton X-100 and centrifuged. The pellets were resuspended in 50 \u0026micro;l PBS containing 1% BSA and 0.5% Tween and 1 \u0026micro;l of primary FITC-labeled anti-BrdU antibody and kept at 4℃ for 1 h in the dark. After wash with PBS, the cells were incubated with 100 \u0026micro;l RNase A for 30 min at 37℃ and PI for 30 min at 4℃ in the dark.\u003c/p\u003e \u003cp\u003eTumor tissue is cut into pieces and digested with type IV collagenase at 37\u0026deg;C for 1 h. Cells were collected and resuspended with 90 \u0026micro;l buffer. Resuspended cells were incubated with CD11b antibody-coated magnetic beads at 4℃ for 15 min. CD11b\u0026thinsp;+\u0026thinsp;cells were resuspended with PBS and incubated with F4/80 and CD206 antibody for 30 min at 4℃ in the dark, and detected by a flow cytometer.\u003c/p\u003e\n\u003ch3\u003eImmunoprecipitation (IP)\u003c/h3\u003e\n\u003cp\u003eNon-denaturing lysis buffer (Solarbio, Beijing, China) was used to extract total protein, and the concentration of protein was determined by BCA kit. Antibodies were immobilized on AminoLink Coupling Resin (Pierce, USA). The supernatants were incubated with antibody-immobilized resin overnight. Then the resin was washed with elution buffer. The eluted samples were analyzed by immunoblotting. SH3RF2 antibody (sc-100976, Santa Cruz, China), LZTS2 antibody (15677-1-AP, Proteintech, China), MYC antibody (AE070, ABclonal, China), and Flag antibody (AE063, ABclonal, China) was used in this part.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eThe polyacrylamide gel consisted of 5% stacking gel and 8% resolving gel. The protein samples were mixed with loading buffer and then heated in a boiling water bath for 5 min. A volume of 20 \u0026micro;l loading samples was subjected to SDS-PAGE. The separated proteins were transferred to a PVDF membrane. Following sealing with blocking solution, the membrane was incubated with primary antibodies overnight at 4℃ and secondary antibodies for 1 h at 37℃. Finally, ECL luminescence solution was used to develop the blot. β-catenin antibody (51067-2-AP, Proteintech, China), rabbit anti\u0026ndash;goat IgG-HRP (SE238, Solarbio, China), goat anti-rabbit IgG-HRP (SE134, Solarbio, China), goat anti-mouse IgG-HRP (SE131, Solarbio, China), Histone H3 antibody (GTX122148, Gene Tex, USA) and GAPDH antibody (60004-1-Ig, Proteintech, China) were used here. Other antibodies are listed previously.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eReal time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted by chloroform extraction and isopropanol precipitation. NanoDrop One was used to determine the concentration of RNA. RNA is reverse transcribed into cDNA using All-in-One First-Strand SuperMix (Magen Biotechnology, Guangzhou, China). SYBR GREEN were used for real time PCR reaction. The 2 \u003csup\u003e\u0026minus;△△CT\u003c/sup\u003e method was used to analyze expression data. The forward primers were as follows, SH3RF2: 5\u0026rsquo;-CGTGGTGGTGGAGATGG-3\u0026rsquo;, CCL2: 5\u0026rsquo;-TCATAGCAGCCACCTTCATT-3\u0026rsquo;, CD163: 5\u0026rsquo;-GAGACTGTTAGGGAAGGTG-3\u0026rsquo;, Arg-1: 5\u0026rsquo;-TTTGCTGACATCCCTAAT-3\u0026rsquo;, IL-10: 5\u0026rsquo;-TGAGAACCAAGACCCAGAC-3\u0026rsquo;. The reverse primers were as follows, SH3RF2: 5\u0026rsquo;-TGGGAGGTGTAATGTTTGGTG-3\u0026rsquo;, CCL2: 5\u0026rsquo;-TCACAGCTTCTTTGGGACAC-3\u0026rsquo;, CD163: 5\u0026rsquo;-TGTTTGTTGCCTGGATT-3\u0026rsquo;, Arg-1: 5\u0026rsquo;-TTCCGTTCTTCTTGACTT-3\u0026rsquo;, IL-10: 5\u0026rsquo;-CATTCTTCACCTGCTCCAC-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTOPflash/FOPflash reporter assay\u003c/h2\u003e \u003cp\u003eTOPflash/FOPflash vector was purchased from YouBio (Shanghai, China). To verify the effect of SH3RF2 on the activation of β-catenin signal pathway, TOP flash (or FOPflash) vector and pRL-TK vector were co-transfected into SH3RF2 stably overexpressed or knocked down LUSC cells using Lipofectamine 3000. A microplate reader was used to detect luciferase activity. The activity of firefly luciferase was normalized to that of renilla luciferase.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as mean with SD and analyzed by GraphPad Prism. Data with normal distribution and homogeneity of variance were analyzed by unpaired t test or one- or two-way ANOVA. Tukey\u0026rsquo;s post hoc test was applied to test multiple comparisons. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was regarded as significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSH3RF2 is upregulated in LUSC patients and is associated with poor survival rate\u003c/h2\u003e \u003cp\u003eTo explore the underlying mechanism of LUSC, we downloaded the gene expression data of lung tissue of LUSC patients from GEO (GSE33532 and GSE19188) and TCGA database. Based on the criteria of p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and ∣log2FC∣\u0026gt;1, the differentially expressed genes (DEGs) in the three datasets were presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. There are 848 up-regulated DEGs and 1189 down-regulated DEGs shared in these three datasets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). GO and KEGG analysis were performed on these DEGs. Top 20 GO terms and top 15 KEGG pathways according to the p-value were respectively presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD. Among the shared DEGs, SH3RF2, a gene that interests us, was screened out. The expression analysis of SH3RF2 in GSE33532, GSE19188, TCGA and GEPIA showed that SH3RF2 was specifically up-regulated in LUSC patients. However, it did not show an up-regulation trend in LUAD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Kaplan Meier plot showed that SH3RF2 has a stronger association with poor survival in LUSC patients compared with LUAD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Therefore, we selected SH3RF2 for the further study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSH3RF2 promotes the proliferation of LUSC cells\u003c/h2\u003e \u003cp\u003eSH3RF2 knockdown and overexpression were conducted in NCI-H226 and SKMES1 cells. SH3RF2 knockdown decreased the colony formation rate and its overexpression increased the colony formation rate (Supplementary Fig.\u0026nbsp;1A). The cell viability was inhibited in the SH3RF2 knockdown group and promoted in the SH3RF2 overexpression group (Supplementary Fig.\u0026nbsp;1B). Compared with the NC\u003csup\u003esh\u003c/sup\u003e group, the proportion of G1 phase cells increased and the proportion of S and G2 phases decreased in SH3RF2 knockdown group. The overexpression group showed the opposite trend (Supplementary Fig.\u0026nbsp;1C). All above demonstrated the positive effect of SH3RF2 on the proliferation of LUSC cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSH3RF2 promotes CCL2 secretion and M2 polarization\u003c/h2\u003e \u003cp\u003eThe RNA level of CCL2 was decreased after SH3RF2 downregulation and increased after SH3RF2 upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We also evaluated the level of CCL2 in the cell supernatant and found that upregulation of SH3RF2 increased the protein level of CCL2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To explore the effect of SH3RF2 on M2 polarization, human THP-1 cells were treated with 150 nM PMA to induce the differentiation into M0 macrophages. The conditioned medium (CM) from NCI-H226 and SKMES1 cells were used to culture the M0 cells for 24 h. Transwell assay showed that SH3RF2 played a promoting role in the migration and invasion ability of M0 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The RNA levels of Arg-1, CD163 and IL-10 were decreased in M0 cells of the SH3RF2 knockdown group and increased in M0 cells of the SH3RF2 overexpression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). After culture using CM, the proportion of CD206\u0026thinsp;+\u0026thinsp;cells contained in M0 cells in the knockdown group decreased, while that in the overexpression group increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSH3RF2 regulates β-catenin in the LUSC cells\u003c/h2\u003e \u003cp\u003eUpregulation and downregulation of SH3RF2 did not affect the total protein level of β-catenin in LUSC cells, but affects its protein level in the nucleus (Supplementary Fig.\u0026nbsp;2A). Immunofluorescence staining of β-catenin (red) showed that SH3RF2 overexpression increased the red fluorescence intensity in the nucleus (Supplementary Fig.\u0026nbsp;2B). The TOPflash/FOPflash luciferase reporter system was used to measure the activation of β-catenin signal pathway. FOPflash, a mutant TOPflash was used as a control. Cells with SH3RF2 knockdown showed a reduced luciferase activity, and cells with SH3RF2 overexpression showed an elevated luciferase activity (Supplementary Fig.\u0026nbsp;2C). SH3RF2 promoted the activation of β-catenin signal pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSH3RF2 affects the development of tumor cells and macrophages through β-catenin\u003c/h2\u003e \u003cp\u003eICG-001, the inhibitor of β-catenin/TCF mediated transcription, was used to treat SKMES1 cells for 24 h. SH3RF2 overexpression improved the cell viability, while ICG-001 reversed this promoting effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). After treatment of ICG-001, the cells were cultured in normal medium for 24 h. The levels of CCL2 in cells and cell supernatant were determined by real time PCR and ELISA respectively. It found that the expression of CCL2 was promoted by SH3RF2 and inhibited by ICG-001 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C). The conditioned medium of the above cells was used to treat M0 cells. ICG- 001 alleviated the positive effect of SH3RF2 on cell migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The RNA levels of Arg-1, CD163 and IL-10 in M0 cells were increased in SH3RF2 overexpression group, and decreased after ICG-001 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In addition, the proportion of CD206\u0026thinsp;+\u0026thinsp;cells showed a same trend in the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). It demonstrated that ICG-001 inhibit the process regulated by SH3RF2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEffect of SH3RF2 on the growth of LUSC tumor\u003c/h2\u003e \u003cp\u003eSKMES1 cells with SH3RF2 silence and overexpression were injected into nude mice. The representative pictures of tumor were presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B. Tumor volume and weight were decreased in the SH3RF2 knockdown group and increased in the SH3RF2 overexpression group. The levels of CCL2 in tumor tissues was elevated by SH3RF2 upregulation and reduced by SH3RF2 downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). Immunohistochemical staining analyzed the expression of SH3RF2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and Ki67 in tumor tissues. The positive staining of Ki67 was enhanced in the SH3RF2 overexpression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Flow cytometry found that the proportion of CD11b\u0026thinsp;+\u0026thinsp;CD206\u0026thinsp;+\u0026thinsp;F4/80\u0026thinsp;+\u0026thinsp;cells was decreased after SH3RF2 downregulation and increased after SH3RF2 upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eTarget protein of SH3RF2\u003c/h2\u003e \u003cp\u003eWe obtained a PPI network of SH3RF2 with its potential target proteins from the BioGRID database (Supplementary Fig.\u0026nbsp;3A). Among these candidate proteins, we noticed the protein LZTS2, which was reported to regulated the β-catenin. Therefore, co-IP was performed in SKMES1 and NCI-H226 cells and found that SH3RF2 interacted with LZTS2 (Supplementary Fig.\u0026nbsp;3B).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eSH3RF2 regulates the ubiquitination of LZTS2\u003c/h2\u003e \u003cp\u003eThe protein level of LZTS2 was increased in cells with SH3RF2 knockdown and decreased in cells with SH3RF2 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). After the LUSC cells were treated with 20 \u0026micro;g/ml cycloheximide (CHX) for 0, 30, 60, 120 and 240 min, the protein levels of LZTS2 in the SH3RF2 overexpression group gradually decreased with increasing CHX treatment time (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D). SKMES1 cells with SH3RF2 overexpression were transfected with MYC-LZTS2 vector and HA-Ub vector. After 42 h, the cells were treated with 10 \u0026micro;M MG132 for 6 h. Compared with the EV group, the ubiquitination of LZTS2 in the SH3RF2 overexpression group was enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). To confirm whether the RING domain of SH3RF2 is involved in the regulation of LZTS2 ubiquitination, we mutated the C28S, C33S and C36S sites of SH3RF2 (RING domain inactivation) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Co-IP found that ubiquitination levels of LZTS2 were reduced in the SH3RF2 mutant group compared to wild-type SH3RF2 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eSH3RF2 regulates LUSC cell growth, macrophage chemotaxis and M2 polarization through LZTS2\u003c/h2\u003e \u003cp\u003eLZTS2 was overexpressed in the SKMES1 cells with SH3RF2 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The cell viability of cells with SH3RF2 and LZTS2 overexpression were decreased compared with the cells with SH3RF2 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Immunofluorescence for β-catenin showed that SH3RF2 promoted the nuclear translocation of β-catenin, while overexpression of LZTS2 alleviated this promotion effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The protein level of CCL2 and the RNA levels of Arg-1, CD163 and IL-10 were decreased in the cells with SH3RF2 and LZTS2 overexpression compared with the cells with SH3RF2 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and E). Transwell assay found that the number of migrated M0 cells in the LZTS2 upregulation group reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). In the same, the proportion of CD206\u0026thinsp;+\u0026thinsp;cells decreased in the LZTS2 upregulation group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe impact of SH3RF2 inhibition and radiation therapy in the growth of LUSC.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the group with SH3RF2 knockdown and radiotherapy treatment, the tumor volume and weight were smaller than those in the group with SH3RF2 knockdown alone (Supplementary Fig.\u0026nbsp;4A). In addition, the immunohistochemistry staining for Ki-67 revealed that the positive staining was inhibited in the cells with SH3RF2 knockdown and radiotherapy treatment (Supplementary Fig.\u0026nbsp;4B).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study clarified the promoting effect of SH3RF2 on LUSC proliferation and growth, expression of chemokine CCL2, and M2-type polarization. We also revealed the SH3RF2-targeted protein LZTS2 and the ubiquitination of LZTS2 through the RING domain of SH3RF2. Collectively, our results clarified the relationship between SH3RF2-LZTS2-β-catenin axis in LUSC. Unfortunately, this study was unable to collect LUSC patient samples to verify the correlation between SH3RF expression and clinically relevant indicators.\u003c/p\u003e \u003cp\u003eCurrently, there are few studies on SH3RF2. In several cancer-related reports, it has been shown to promote tumor progression. Our research results also confirmed its cancer-promoting effect in LUSC. SH3RF2, as an E3 ubiquitin ligase, has been shown to regulate the ubiquitination of related proteins. SH3RF2 can degrade POSH protein through ubiquitination and the RING domain of SH3RF2 and the RING domain of POSH are involved in this degradation process (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). SH3RF2 directly binds ACLY and promotes its K48-linked ubiquitination-dependent degradation (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). SH3RF2 promotes K48-linked ubiquitination of RBPMS, thereby increasing its proteasomal degradation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In this study, we found that SH3RF2 regulated the ubiquitination of LZTS2 through its RING domain. However, these studies including the current study only revealed that SH3RF2 is involved in ubiquitination, but did not clarify the specific way or binding region of SH3RF2 binding to target proteins. A report demonstrated that the third SH3 domain (370\u0026ndash;459 amino acids) of SH3RF2 protein is a specific PAK4 binding region and inhibits PAK4 ubiquitination, resulting in the upregulation of PAK4 protein (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Although this report revealed the specific domain of SH3RF2 that binds target proteins, the difference is that SH3RF2 inhibits the ubiquitination of target proteins through this domain, which is significantly different from those studies mentioned above. All of these results reflect that the three SH3 domains and the RING domain contained in SH3RF2 may have special functions and significance. As far as the RING domain alone is concerned, it is involved in the ubiquitination process. The SH3 domain is reported to bind to its target partner with a typical PXXP motif (X represents any amino acid), and has functions such as regulating endocytosis and actin cytoskeleton remodeling (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In the SH3RF2 protein, what functions the combination of these domains can play is worthy of further exploration.\u003c/p\u003e \u003cp\u003eIt is well known that macrophage M1/M2 polarization is important to the development of cancer. M2 macrophage is also known as tumor-associated macrophage, which can promote tumor growth, invasion, metastasis and angiogenesis, and inhibit tumor immune response (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In non-small cell lung cancer, M2 macrophages have been shown to promote tumor growth and migration (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). M2 macrophages mainly promote tumor occurrence and metastasis by secreting angiogenic factors, chemokines, and downregulating major histocompatibility complex class II (MHC II) and costimulatory ligands CD80 and CD86 (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Here, we found that SH3RF2 could promote M2 polarization and increase the proportion of M2 macrophages. In addition, our results found that SH3RF2 inhibited the expression of CCL2 in tumor cells. CCL2 is known to promote M2 polarization. It indirectly reflects the promotion effect of SH3RF2 on M2 polarization. It is worth mentioning that reducing the proportion of M2 macrophages is gradually recognized as a new strategy for tumor immunotherapy (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Therefore, our results provide new candidate targets for immunotherapy of LUSC.\u003c/p\u003e \u003cp\u003eThe Wnt/β-catenin pathway is increasingly recognized as a potentially important target for anticancer therapy (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Nuclear translocated β-catenin activates genes related to cell proliferation, survival, differentiation, and migration through TCF/LEF transcription factors (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In various tumors such as colorectal cancer (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), prostate cancer (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), and lung cancer (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), LZTS2 has been reported to be involved in the activation of the Wnt/β-catenin pathway. We found that SH3RF2 promoted the nuclear import of β-catenin, and ICG-001 alleviated the promoting effects of SH3RF2 overexpression on tumor cell proliferation, macrophage chemotaxis and M2 polarization. In addition, SH3RF2 regulated the ubiquitination of LZTS2. Rescue experiment demonstrated that SH3RF2 regulates LUSC cell growth, macrophage chemotaxis and M2 polarization through LZTS2. Obviously, these effects can be achieved through the SH3RF2-LZTS2-β-catenin cascade reaction.\u003c/p\u003e \u003cp\u003eCompared with LUAD patients, patients with advanced LUSC had higher ORR, shorter PFS and OS rates in first-line immunotherapy (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Since LUSC has very few driver gene mutations and only 3% of patients have epidermal growth factor receptor mutations and anaplastic lymphoma kinase rearrangements, radiotherapy is more important for the treatment of LUSC than LUAD (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Most patients with early-stage LUSC can be effectively treated with radiation therapy, but patients with advanced LUSC may have limited radiotherapy treatment (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Therefore, in order to improve the survival rate of LUSC, developing new treatment strategies is a top priority. We tested the effect of combined treatment with radiotherapy and SH3RF2 overexpression on xenografted tumors and found that it could significantly inhibit tumor growth. This may provide new directions for developing new treatment strategies.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthical Approval\u003c/strong\u003e \u003cp\u003eAll animal experiments were approved by the Laboratory Animal Ethics Committee of the Fourth Hospital of Hebei Medical University (No. IACUC-4th Hos Hebmu) and performed in compliance with ARRIVE guidelines.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by the Natural Science Foundation of Hebei Province (No. H2023206301).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJie Yang: Funding acquisition, investigation and methodology. Zhong-Fei Jia: Investigation and data curation. Juan Li: Investigation. Chao Jiang: Formal analysis. Xin Zhao, Yu-Xiang Wang and Xiao-Guo Ma: visualization; writing\u0026ndash;original draft. Xin-Jian Xu: Conceptualization and project administration. All authors reviewed and edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAll authors thank the Fourth Hospital of Hebei Medical University and the Natural Science Foundation of Hebei Province for their assistance.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDempke WC, Suto T, Reck M. Targeted therapies for non-small cell lung cancer. Lung Cancer. 2010;67(3):257\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerbst RS, Morgensztern D, Boshoff C. The biology and management of non-small cell lung cancer. 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Sh3rf2/POSHER protein promotes cell survival by ring-mediated proteasomal degradation of the c-Jun N-terminal kinase scaffold POSH (Plenty of SH3s) protein. J Biol Chem. 2012;287(3):2247\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang X, Sun D, Xiang H, Wang S, Huang Y, Li L, et al. Hepatocyte SH3RF2 Deficiency Is a Key Aggravator for NAFLD. Hepatology. 2021;74(3):1319\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBogic L, Gerlach JL, McEwen BS. The ontogeny of sex differences in estrogen-induced progesterone receptors in rat brain. Endocrinology. 1988;122(6):2735\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoussens LM, Zitvogel L, Palucka AK. Neutralizing tumor-promoting chronic inflammation: a magic bullet? Science. 2013;339(6117):286\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Li H, Wu Q, Chen Y, Deng Y, Yang Z, et al. Tumoral NOX4 recruits M2 tumor-associated macrophages via ROS/PI3K signaling-dependent various cytokine production to promote NSCLC growth. Redox Biol. 2019;22:101116.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu CS, Shiau AL, Su BH, Hsu TS, Wang CT, Su YC, et al. Oct4 promotes M2 macrophage polarization through upregulation of macrophage colony-stimulating factor in lung cancer. J Hematol Oncol. 2020;13(1):62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadeddu C, Donisi C, Liscia N, Lai E, Scartozzi M, Maccio A. EGFR-Mutated Non-Small Cell Lung Cancer and Resistance to Immunotherapy: Role of the Tumor Microenvironment. Int J Mol Sci. 2022;23(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Xie F, Li K, Zhang H, Yin Y, Yu Y, et al. Gold nanoparticle-directed autophagy intervention for antitumor immunotherapy via inhibiting tumor-associated macrophage M2 polarization. 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LZTS2 and PTEN collaboratively regulate ss-catenin in prostatic tumorigenesis. PLoS ONE. 2017;12(3):e0174357.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin J, Yi S, Zhou H, Zeng C, Zou M, Zeng X, et al. Efficacy of radiotherapy in combination with first-line immunotherapy and chemotherapy for advanced lung squamous cell carcinoma: a propensity score analysis. Front Immunol. 2023;14:1138025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantivasi WL, Xia F. Ionizing radiation-induced DNA damage, response, and repair. Antioxid Redox Signal. 2014;21(2):251\u0026ndash;9.\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biology-direct","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bdir","sideBox":"Learn more about [Biology Direct](http://biologydirect.biomedcentral.com)","snPcode":"13062","submissionUrl":"https://submission.nature.com/new-submission/13062/3","title":"Biology Direct","twitterHandle":"@Biology_Direct","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lung squamous cell carcinoma, SH3RF2, LZTS2, β-catenin, M2 polarization","lastPublishedDoi":"10.21203/rs.3.rs-5457209/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5457209/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Although the treatment of lung cancer has been well developed, the survival rate for lung squamous cell carcinoma (LUSC) is still low. It is meaningful to explore new molecular targets and develop new treatment strategies. SH3RF2 is an E3 ubiquitin ligase containing 3 SH3 domains and has not been reported in LUSC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eSH3RF2 promoted the proliferation of LUSC cells, and the nuclear translocation of β-catenin, increased Arg-1, CD163 and IL-10 RNA levels and the proportion of CD206+ cells in M0 THP-1 cells, and enhanced the migration and invasion of M0 THP-1 cells. ICG-001 alleviated the above effects of SH3RF2 on M0 THP-1 cell. In vivo tumorigenesis experiments found that SH3RF2 promoted tumor growth and increased the proportion of M2 cells. IP found that SH3RF2 interacted with LZTS2 and regulated the ubiquitination of LZTS2 with RING domain. LZTS2 overexpression reduced the nuclear translocation of β-catenin, cell migration and invasion, and M2 polarization promoted by SH3RF2 overexpression. The combination of SH3RF2 overexpression and radiotherapy inhibited the growth of tumor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e This study elucidates the cancer-promoting role of SH3RF2, its positive effect on M2 polarization, and its relationship with LZTS2 and β-catenin. It provides new candidate molecular targets for the treatment of LUSC.\u003c/p\u003e","manuscriptTitle":"The role of SH3RF2 in lung squamous cell carcinoma and M2 polarization: insights into LZTS2 ubiquitination","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-03 16:11:48","doi":"10.21203/rs.3.rs-5457209/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-24T21:12:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-24T15:27:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100747336070353504875469711893077417799","date":"2025-03-10T09:24:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"23002403337425691764799643808130352471","date":"2025-02-08T15:17:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-07T03:59:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92050482462175463624536128809493178217","date":"2024-12-17T10:02:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-05T09:18:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-26T07:48:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-19T13:25:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biology Direct","date":"2024-11-15T03:06:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biology-direct","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bdir","sideBox":"Learn more about [Biology Direct](http://biologydirect.biomedcentral.com)","snPcode":"13062","submissionUrl":"https://submission.nature.com/new-submission/13062/3","title":"Biology Direct","twitterHandle":"@Biology_Direct","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"93718658-cd55-416e-bcfc-a41c982ed2fd","owner":[],"postedDate":"December 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-07T07:11:52+00:00","versionOfRecord":{"articleIdentity":"rs-5457209","link":"https://doi.org/10.1186/s13062-025-00677-0","journal":{"identity":"biology-direct","isVorOnly":false,"title":"Biology Direct"},"publishedOn":"2025-07-17 16:05:20","publishedOnDateReadable":"July 17th, 2025"},"versionCreatedAt":"2024-12-03 16:11:48","video":"","vorDoi":"10.1186/s13062-025-00677-0","vorDoiUrl":"https://doi.org/10.1186/s13062-025-00677-0","workflowStages":[]},"version":"v1","identity":"rs-5457209","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5457209","identity":"rs-5457209","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}