KDM3B promotes neural invasion in colorectal cancer through TrkA upregulation by inhibiting H3K9 dimethylation

KDM3B promotes neural invasion in colorectal cancer through TrkA upregulation by inhibiting H3K9 dimethylation | 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 Article KDM3B promotes neural invasion in colorectal cancer through TrkA upregulation by inhibiting H3K9 dimethylation Juan Li, Sizheng Sun, Xing Wang, Yinchun Li, Huifeng Zhang, Xianhua Fu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6568594/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Neural invasion (NI) represents a critical factor contributing to the unfavorable prognosis of patients diagnosed with colorectal cancer (CRC), necessitating an investigation into its underlying mechanisms. This study identifies lysine demethylase 3B (KDM3B) as a pivotal protein associated with NI in CRC, as determined through tandem mass tag sequencing (TMT-Seq) analysis of clinical CRC tissues, which involved a comparative assessment of control and NI samples. Furthermore, Cut-tag and ATAC-Seq experiments conducted on CRC cell lines (comparing control to sh-KDM3B) demonstrated that KDM3B enhances the expression of the nerve growth factor receptor NTRK1, which encodes the TrkA protein in CRC. This finding was corroborated through both in vivo and in vitro experiments. The results indicated that KDM3B overexpression in CRC results in the inhibition of H3K9me2, which subsequently leads to the upregulation of TrkA. This upregulation facilitates the binding of nerve growth factor (NGF) to TrkA, thereby promoting NI in CRC. This study elucidates the intrinsic mechanisms underlying NI in CRC and provides a robust theoretical framework for considering KDM3B as a prognostic indicator and a potential therapeutic target for the disease Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Computational biology and bioinformatics Biological sciences/Genetics Biological sciences/Molecular biology Neural invasion (NI) Colorectal cancer (CRC) Lysine demethylase 3B (KDM3B) H3K9 dimethylation (H3K9me2) Tyrosine kinase receptor A (TrkA) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Colorectal cancer (CRC), as the third most common cancer in the world with a mortality rate ranking the third, poses a serious threat to human health[ 1 ]. Metastasis is a key risk factor for poor prognosis in patients with CRC. The 5-year survival rate of metastatic CRC is less than 15% [ 2 ], and neural invasion (NI) is a major risk factor for local recurrence and distant metastasis of CRC[ 3 ]. Systemic and local cross-linking between the nervous system and tumors has become a new hotspot in the tumor control research[ 4 ]. In CRC, NI can drive the tumor neovascularization and induce the tumor metastasis[ 5 ]. Besides, nervous system disorders can also affect the immune system and promote the neoplastic tumorigenesis[ 6 ]. Understanding the mechanism of NI in CRC and inhibiting its occurrence can help improve the survival rate and quality of life of patients. Neurotrophic receptors are expressed on the surface of various tumor cells, with common ones including tyrosine kinase receptor A (TrkA), tyrosine kinase receptor B (TrkB), and tyrosine kinase receptor C (TrkC) encoded by the NTRK gene. Nerve growth factor (NGF) binds to TrkA[ 7 , 8 ], brain-derived neurotrophic factor (BDNF) and neurotrophic factor 4 (NT-4) both bind to TrkB [ 9 – 11 ], and neurotrophic factor 3 (NT-3) binds to TrkC[ 12 ]. Different neurotrophic receptors tend to exhibit different physiological processes after binding to neurotrophic factors. In tumors, there are abnormalities in NTRK gene expression[ 13 ], which causes the overexpression of neurotrophic receptors on the tumor cell surface. Meanwhile, excessive binding of neurotrophic receptors to neurotrophic factors can activate the MAPK, PI3K and PLCγ pathways in tumors[ 14 , 15 ], thereby inducing the malignant tumor progression, which is especially critical in the NI process [ 16 ]. Hence, reducing the binding between neurotrophic receptors and factors becomes crucial to mitigate the NI in CRC. DNA methylation, as an important mode of epigenetic modification, occurs predominantly on cytosine in cytosine-phosphate-guanine (CpG) dinucleotides[ 17 ]. Abnormal DNA methylation plays a pivotal role in tumor biology, which causes the silencing of some tumor suppressor genes, the activation of proto-oncogenes, and the abnormal expression of transposons, increasing genomic instability and creating favorable conditions for tumorigenesis and development[ 18 – 21 ]. The in-depth study of tumor methylation not only facilitates better understanding of the tumor pathogenesis, but also provides new ideas and directions for early diagnosis, prognostic assessment and targeted therapy of tumors. In this study, through proteomic sequencing on the clinical tissues of CRC patients with or without NI, we detected the highly expressed protein KDM3B in neuroinvasive CRC tissues. As a histone demethylase, lysine demethylase 3B (KDM3B) often acts on H3K9me1 and H3K9me2[ 22 ]. The downstream sequence NTRK1 of KDM3B action on H3K9me1 and H3K9me2, as well as its chromatin openness in CRC cells before and after KDM3B knockdown were detected by CHIP-Seq and ATAC-Seq. TrkA was identified as the downstream site of KDM3B's role in CRC, which was then verified through in vivo and in vitro experiments. The present study reveals the mechanism whereby KDM3B promotes NI in CRC by the H3K9me2 demethylation-mediated upregulation of TrkA, laying a solid theoretical foundation for the application of KDM3B as a prognostic indicator and therapeutic target for CRC (Graphical abstract). Materials and methods Subject inclusion and exclusion criteria The clinical part of this study was approved by the Ethics Committee of The Affiliated Taizhou People's Hospital of Nanjing Medical University (KY2023-164-01). Surgically resected tumor tissues from six CRC patients, three without NI and three with NI, were collected for the follow-up study. Inclusion criteria: 1. CRC confirmed by colonoscopy and pathological biopsy; 2. Patients aged between 18 and 75 years; 3. Newly diagnosed patients who have not yet received any anticancer therapy; 4. Eastern Cooperative Oncology Group (ECOG) score of 0-2 points; 5. After being fully informed of the research purpose, method, process, potential risks and benefits, patients or their legal representatives voluntarily signed informed consent and agreed to participate in this clinical study. Exclusion criteria: 1. Complicated with cardiovascular and cerebrovascular diseases, severe hepatic and renal insufficiency, other serious diseases such as malignant tumors; 2. Mental or cognitive impairment; 3. Patients with poor compliance; 4. Received a specific treatment or trial within 3 months. Cell culture and transfection The STR-validated ovarian cancer cell lines HCT116 and HT29, which were procured from Procell Life Sciences (China), were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). To silence lysine demethylase 3B (KDM3B), we transfected the HCT116 and HT29 cells with lentivirus sh-KDM3B and sh-NTRK1 plasmids that were chemically synthesized by GenePharma (Shanghai, China). The sequences of sh-KDM3B were as follows: sh-KDM3B#1: 5'-CCGATGTGAAAGTTCGGGATT-3'; sh-KDM3B#2: 5'-GCGATCTTTGTAGAATTTGAT-3'; sh-KDM3B#3: 5'-GTTAGTCTTCCTTTCGATAAA-3'. The sequences of sh-NTRK1 were as follows: sh-NTRK1#1: 5'-TATCTACAGCACCGACTATTA-3'; sh-NTRK1#2: 5'-TGCCTTCATGGACAACCCTTT-3'; sh-NTRK1#3: 5'-CATCGAGAACCCACAATACTT-3'. Cell migration and invasion assays To assess the migration ability of cells, we created a linear wound on each nearly fused monolayer of HCT116 and HT29 cells. Images were acquired at 0, 24, and 48 h after scratching to measure the scratch closure for quantifying cell migration. The invasion ability of the cells was detected by Transwell assay. The Matrigel-coated upper chamber of Transwell inserts was added with 1×10 4 HCT116 and HT29 cells, while the lower chamber was supplemented with a culture medium containing 20% FBS. Three d later, non-migrated cells were removed, and the migrated cells were fixed, stained, counted, and photographed under an inverted microscope (Nikon, Japan). DRG tumor-cell co-culture assay A total of 2×10 5 CRC cell lines, HCT116 and HT29, were suspended in 25 µL of growth factor-reduced Matrigel (Sigma-Aldrich, USA) and placed in the center of each of the 12 wells. The dorsal root ganglia (DRGs) of each 8-d-old Sprague-Dawley (SD) rats were extracted, and inoculated with 25 µL of Matrigel near the cell suspension, while an additional 25 µL of blank Matrigel was added on the opposite side (Figure 2.D) [23]. The Petri dishes were incubated at 37°C in CO 2 with 5% humidity for 30 min to allow solidification. Thereafter, the cultures were incubated with 10% FBS-containing NeuroBasal medium (Invitrogen, USA). The degree of tumor NI was observed microscopically at d 0, 3, and 7, and images were collected. Western Blot Analysis Total proteins were extracted from HCT116 and HT29 cells using RIPA lysis buffer containing phenylmethylsulfonyl fluoride (Beyotime, USA). After quantification with BCA protein assay kit, equal amounts of proteins were separated by 10% SDS-PAGE and then transferred onto PVDF membranes. Subsequently, the membranes were blocked in 5% skim milk powder at room temperature for 2 h, and then incubated with primary antibodies against KDM3B (MA5-32123; Thermo Fisher Scientific, USA) and TrkA (PA5-17170; Thermo Fisher Scientific, USA) overnight at 4°C. Finally, the membranes were incubated with HRP-conjugated secondary antibody at room temperature for 2 h, followed by the observation of chemiluminescent signals using Pierce™ ECL substrates (Thermo Fisher Scientific, USA). mRNA sequencing (mRNA-Seq) mRNA-Seq was accomplished by LC-Bio Technologies (Hangzhou) Co., Ltd. The cells were lysed in Trizol (Takara, Japan), total RNA was prepared using RNA Clean & Concentrator-5 ™ (Zymo, USA), and genomic DNA contamination was eliminated using DNase. After isolating with the NEBNext PolyA mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, USA), the mRNA was used for RNA-Seq library preparation by the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, USA). The libraries were sequenced in Illumina with the paired-end 2 x 150 sequencing mode. Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-Seq) A total of 50,000 viable cells were collected, washed in cold PBS and centrifuged at 500 g and 4°C for 5 min. Each sample was resuspended with 50 μl of lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 0.1 % IGEPAL, 0.1 % NP-40, 0.1 % Tween-20) and then incubated on ice for 10 min. The supernatant was discarded after 5 min of centrifugation at 500 g and 4 °C, while the cell nuclei were collected for subsequent transposition reaction. Library construction was performed following the instructions of TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, China). Finally, the libraries were sequenced on the Illumina Novaseq 6000 platform at 150 bp paired-end read lengths by Genefund Biotech (Shanghai). Cleavage under targets and tagmentation (CUT&Tag) CUT&Tag assays were conducted on fresh WDLPS, DDLPS, and PA cells (100,000 cells/sample), treated with or without 0.8 μM QN-302, for 4 h. After collection and washing with wash buffer, the cells were immobilized on concanavalin A-coated magnetic beads (Bangs Laboratories Inc., USA) and incubated overnight at 4°C with 500 ng FLAG-tagged anti-G4 BG4 antibody. Then, the samples were incubated at RT for 1 h with anti-FLAG antibody (Merck, USA), and further with rabbit anti-mouse IgG (Merck, USA). Bead-bound cells were washed in dig-wash buffer, bound with pA–Tn5 adapter complex, and tagmented at 37°C for 1 h. After the tagmentation, libraries were purified by phenol–chloroform extraction and amplified using NEBNext High-Fidelity 2× PCR Master Mix with barcoded i5 and i7 primers. CUT&Tag libraries were size-selected with Agencourt AMPure XP Beads (Beckman Coulter, USA) and size-analyzed by Bioanalyzer (Agilent Technologies, Italy). Finally, the samples were sequenced on an Illumina NextSeq500 platform with paired-end 38 bp reads. In vivo NI model The in vivo NI model was established by a modified method based on a previously described protocol [24]. Four-weeks-old male nude mice were anesthetized by methoxyflurane inhalation for subsequent surgery. The sciatic nerve was surgically exposed and 5 μL of 2×10 5 /μL CRC cell suspension was injected around it. To assess the effect of KDM3B knockdown on NI, the tumor volume as well as sciatic nerve-related hindlimb behavior were examined every 7 d. Four weeks later, the mice were euthanized and the xenografts were excised to compare tumor size. This work has been reported in accordance with the ARRIVE guidelines[25]. Statistical analysis Data from three independent experiments were expressed as means ± standard deviations. Differences between two groups were analyzed by paired or unpaired Student's t-test, while differences among multiple groups were evaluated by one-way ANOVA and Tukey's test. The correlation between gene expression data was tested with Pearson's correlation coefficient. P < 0.05 was considered statistically significant. Results Proteomic screening of KDM3B, a key protein inducing NI in CRC To explore the key proteins responsible for inducing NI in CRC, we clinically collected surgical tumor tissues from six patients with pathological stage III CRC complicated by vascular invasion, including three patients in the NI group and three patients in the non-NI group. The tumor tissues from the two groups were subjected to differential protein analysis by TMT-Seq. Compared with the NI group, there were 98 differentially expressed proteins (DEGs) (Fig. 1 .A-B) in the non-NI group, with 27 DEGs located in the cell nuclei (Fig. 1 .C), accounting for the majority, among which the KDM3B protein was the most differentially expressed. As a histone demethylase, KDM3B plays an important role in biological processes such as gene expression regulation, and affects the expression of multiple proteins by changing the methylation level of histones. These DEGs were subjected to GO and KEGG enrichment analyses (Fig. 1 .D-E) for investigating their effects on the CRC functions. GSEA enrichment analyses were separately performed (Fig. 1 .F-G) on the basis of GO and KEGG enrichment. The results showed that the DEGs could be enriched into "GO:0016477 cell migration", "GO:0030335 positive regulation of cell migration", "hsa04010 MAPK signaling pathway", and "hsa04151 PI3K-AKT signaling pathway" that are associated with tumor migration and invasion. The KDM3B levels of the patients were validated by immunohistochemistry. According to our results, the three patients in the NI group all exhibited higher KDM3B levels than the three non-NI patients (Fig. 1 .H). Suggestively, KDM3B plays a crucial role in the NI process of CRC. Binding between neurotrophic receptors and factors was required during NI in CRC To verify whether KDM3B was associated with the migration and invasion of CRC, we first conducted in vitro experiment by knocking down and overexpressing KDM3B in CRC cell lines HCT116 and HT29 (Fig. 2 .A). For the subsequent experiment, sh-KDM3B#2 with the highest knockdown efficiency and the overexpressed oe-KDM3B were selected. Scratch and Transwell assays were applied to examine the cell migration and invasion abilities. In the normal culture environment, the KDM3B level had insignificant effects on the migration and invasion abilities of CRC cells (Fig. 2 .B-C), which was inconsistent with our proteomics results. To further investigate the interaction between CRC cells and nerves, we established a co-culture model of SD rat DRGs and CRC cells that was closer to the in vivo environment (Fig. 2 .D). After 5 d of co-culture between DRGs and CRC cells, the tumor cells began to migrate towards the DRGs, and the DRG axons started to grow towards the CRC cells (Fig. 2 .E). After 7 d of co-culture, we began to compare the migration distance of tumor cells and the growth level of DRGs among the groups. The results showed that the migration distances of HCT116 and HT29 cells were significantly shorter in the sh-KDM3B group than in the control group, while the oe-KDM3B group exhibited significantly longer migration distances than the control group. Comparison of the DRG growth level found that the coverage of DRGs in the sh-KDM3B group was significantly smaller than that of the control group, while the oe-KDM3B group displayed significantly larger coverage than the control group. Suggestively, the DRG growth level was poor in the sh-KDM3B group, whereas was good in the sh-KDM3B group. Excessive binding between the neurotrophic receptors and factors can induce NI in tumors. Changes in both the neurotrophic receptor and factor expressions can affect the degree of tumor NI. By comparing the differences in growth environment between the above experimental tumor cells, we conjectured that the binding between neurotrophic receptors and factors might be involved in the NI process of CRC in synergy with KDM3B. By adding the neurotrophic factors on the basis of the previous experimental conditions, we reexamined the migration and invasion abilities of CRC cells through scratch and Transwell assays (Fig. 2 .F-G). It was found that under the synergy of neurotrophic factors, the migration and invasion abilities were significantly weaker in the sh-KDM3B group than in the control group, while the oe-KDM3B group exhibited significantly stronger migration and invasion abilities than the control group. Thus, KDM3B requires the binding of neurotrophic receptors to neurotrophic factors in the process of inducing CRC NI. KDM3B induced CRC NI by triggering TrkA overexpression To investigate the specific reasons for the elevated degree of neurotrophic receptor and factor binding in CRC patients with NI, we first performed transcriptomic sequencing on the HCT116 and HT29 CRC cell lines treated with sh-vector and sh-KDM3B, finding a total of 2,021 common DEGs (Fig. 3 .A). These DEGs were subjected to the GO and KEGG enrichment analyses (Fig. 3 .B), and the results showed that they could be enriched into such pathways as "GO:0016477 RNA splicing", "GO:0030335 nuclear transport", and "GO:0030335 protein localization to nuclear body", which are associated with the nuclear entry and role of proteins. In view of the entry and role of KDM3B in the cell nuclei, we next explored the differences in KDM3B-binding sequences by performing Cleavage Under Targets and Tagmentation (Cut-tag) on the sh-NC and sh-KDM3B-treated CRC cell lines HCT116 and HT29 (Fig. 3 .C). A total of 2,860 inter-group differentially expressed KDM3B-binding DNA sequences were identified, most of which were promoter sequences (Fig. 3 .D). Through the GO and KEGG enrichment analyses of the inter-group differentially expressed sequences (Fig. 3 .E), we found that the DEGs could be enriched into such pathways as "GO:0001841 neural tube formation". "GO:0002467 germinal center formation", and "hsa:04390 Hippo signaling pathway", which are associated with the tumor NI. We further examined the chromatin accessibility of sh-vector and sh-KDM3B-treated HCT116 and HT29 cells by ATAC-Seq assay (Fig. 3 .F). A total of 2,749 inter-group differentially expressed sequences of chromatin openness were identified, most of which were promoter sequences localized near the transcription start sites (TSS) (Fig. 3 .G). According to the GO and KEGG enrichment analyses of inter-group DEGs obtained after the ATAC-Seq analysis (Fig. 3 .H), the DEGs could be enriched into such pathways as "GO:0010975 regulation of neuron projection development", "hsa:04015 Rap1 signaling pathway", and "hsa:04520 Adherens junction", which are related to nerve growth. Thus, KDM3B is somewhat associated with NI in CRC cells. We intersected the DEGs obtained from mRNA-Seq, Cut-tag and ATAC-Seq analyses to identify a total of 155 common DEGs (Fig. 3 .H). Among them, TrkA-encoding NTRK1 was significantly differentially expressed in all of the mRNA-Seq, Cut-tag and ATAC-Seq (Fig. 3 .J). Therefore, the level of NTRK1 involved in transcription was downregulated after KDM3B knockdown, and at the same time, the chromatin of NTRK1 was more difficult to access. Knockdown of KDM3B could inhibit the transcriptional efficiency of NTRK1. NTRK genes encode the neurotrophic receptors TrkA, TrkB, and TrkC, with NTRK1 being the TrkA-encoding gene. Overactivation of NTRK1 could increase the TrkA expression level and induce CRC NI after binding with NGF. Thus, in CRC, KDM3B triggers overexpression of TrkA to induce NI. Further, the above sequencing results were verified by detecting the expression levels of neurotrophic factors and receptors in clinical patients. We collected sera from 60 pathological stage III CRC patients with vascular invasion, including 30 patients in the NI group and 30 patients in the non-NI group. Elisa was applied to examine the differences in the serum levels of NGF (Fig. 3 .K), BDNF (Fig. 3 .L), NT-3 (Fig. 3 .M) and NT-3 (Fig. 3 .N) between the two groups. The results suggested no significant inter-group differences in the NGF, BDNF, NT-3 or NT-3 expression. Immunohistochemistry was performed to assess the differences in the neurotrophic factor expression in the tumor tissues of patients in the two groups (Fig. 3 .O), finding that the TrkA level in the NI group was significantly higher than that in the non-NI group, thus verifying our sequencing results. KDM3B triggered TrkA overexpression by inhibiting H3K9me2 KDM3B, as a histone demethylase, tends to act on H3K9me1 and H3K9me2, thereby affecting the expression of downstream genes and proteins (Fig. 4 .A). We applied Western blot to detect the expression levels of H3K9me1 and H3K9me2 (Fig. 4 .B), finding that the H3K9me2 level in the sh-KDM3B group was significantly lower than that in the control group, while the oe-KDM3B group exhibited significantly higher H3K9me2 level than the control group. No significant inter-group differences were noted in the H3K9me1 level. To further explore the specific mechanism whereby KDM3B promotes the TrkA expression in CRC, we applied Cut-tag to assess the differences in H3K9me1- and H3K9me2-binding sequences in CRC cells before and after the KDM3B knockdown. The results showed that after knocking down KDM3B, there were differences in H3K9me1-binding sequences (Fig. 4 .C), and the inter-group differentially expressed sequences were mostly in the promoter region and localized near the TSS (Fig. 4 .D). The DEGs could be enriched into pathways like "GO:0007059 chromosome segregation", "GO:0006260 DNA replication", and "hsa:04115 p53 signaling pathway" that are closely associated with tumor progression, but failed to be enriched into the tumor NI-related pathways (Fig. 4 .E). Differences in H3K9me2-binding sequences were more significant (Fig. 4 .F), with more number of promoter sequences and denser localization near the TSS (Fig. 4 .G). The DEGs could be enriched into such pathways as "GO:0071897 DNA biosynthetic process", "GO:0006275 regulation of DNA replication", and "hsa:04152 AMPK signaling pathway", which are associated with abnormal biological behaviors of tumors (Fig. 4 .H). By analyzing the NTRK1 level of H3K9me1- and H3K9me2-binding sequences, we found that the NTRK1 level of H3K9me2-binding sequences changed significantly before and after the KDM3B knockdown (Fig. 4 .I). Thus, in CRC, KDM3B is more likely to trigger TrkA overexpression via H3K9me2, ultimately inducing NI. NTRK1 knockdown could reverse the KDM3B-induced NI To further verify our sequencing results, we validated the NI-related phenotypic responses in CRC HCT116 and HT29 cells. Initially, we knocked down and overexpressed TrkA in the CRC cell lines (Fig. 5 .A), and selected sh-NTRK1#1 with the highest knockdown efficiency and the overexpressed oe-NTRK1 for the subsequent experiment. By observing changes in the cell migration and invasion abilities, the NTRK1 level was found to have no significant effect on the migration and invasion abilities of CRC cells in the normal culture environment. After adding neurotrophic factors, the migration and invasion abilities were significantly weaker in the sh-NTRK1 group than in the control group, while the oe-NTRK1 group exhibited significantly stronger migration and invasion abilities than the control group. Overexpression of NTRK1 after KDM3B inhibition partially restored the declined migration and invasion abilities (Fig. 5 .B-C), demonstrating that KDM3B could enhance the migration and invasion abilities of CRC cells by upregulating NTRK1. In the co-culture model of DRGs and CRC cells, the migration distance of tumor cells was significantly shorter in the sh-NTRK1 group than in the control group, while the oe-NTRK1 group displayed significantly longer migration distance than the control group. Overexpression of NTRK1 after KDM3B inhibition could partially restore the reduced neural migration distance. Meanwhile, the coverage of DRGs in the sh-NTRK1 group was significantly smaller than that in the control group, while the oe-NTRK1 group exhibited significantly larger coverage than the control group. Overexpression of NTRK1 after KDM3B inhibition could restore the narrowed nerve coverage. This suggests that NTRK1 can directly affect the degree of NI and the growth level of DRGs in CRC (Fig. 5 .D). The above experiments demonstrated that inhibition of NTRK1 could reverse the NI induction by KDM3B in CRC, thus further verifying that KDM3B can induce CRC NI by triggering the overexpression of TrkA-encoding NTRK1. Validation of in vivo model of CRC NI promoted by KDM3B To further verify our in vitro results, a mouse model of CRC NI was established for in vivo experimental validation. Normal and KDM3B knockdown CRC cells were injected separately around the right and left sciatic nerves of nude mice, during which the bilateral tumor sizes were measured. Four weeks later, the mice were sacrificed, and the degree of bilateral tumor NI was observed by H&E staining. The results suggested that in the nude mouse NI model established by HCT116 and HT29, the right side KDM3B knockdown tumors were significantly smaller than the left side normal tumors (Fig. 6 .A-C), thus proving that KDM3B can promote the growth of CRC. To further investigate the effect of KDM3B on the degree of NI in CRC, H&E staining was performed on bilateral tumors to observe the NI degree. The results showed that in the nude mouse NI model established by HCT116 and HT29, the degree of NI in the right side KDM3B knockdown tumors was significantly lower than that in the left side normal tumors (Fig. 6 .D). Meanwhile, we also applied immunohistochemistry to detect the TrkA expression in bilateral tumors, finding that the TrkA level in the right side KDM3B knockdown tumors was significantly lower than that in the left side normal tumors (Fig. 6 .E). Further, we validated the enriched pathways and found that in the cellular experiments (Fig. 6 .F) and animal experiments (Fig. 6 .G), the overexpression of KDM3B could activate the CRC MAPK and PI3K pathways, thereby inducing NI in CRC. Taken together, both in vivo and in vitro experiments demonstrated that KDM3B could induce CRC NI through upregulation of TrkA-encoding NTRK1 by inhibiting H3K9me2. Discussion As a common demethylase, KDM3B plays a crucial role in multiple biological processes, including the regulation of gene expression[ 22 ]. In tumors, KDM3B often plays a role in the malignant process by regulating the methylation level of histone H3K9[ 26 ]. In our study, proteomics was applied to identify KDM3B, the key inducer protein of NI in CRC. Further, the mechanism for KDM3B-induced CRC NI was explored by CHIP-Seq. It was found that KDM3B promoted the expression of neurotrophic receptor TrkA by inhibiting H3K9me2, thereby promoting the NI in CRC (Graphical abstract). Suggestively, KDM3B can be a potential therapeutic target for CRC NI, which helps improve the prognosis of CRC patients. KDM3B is recognized and bound to nucleosomes containing methylated H3K9 through its specific enzymatically active domain[ 26 ]. Then, utilizing its catalytically active center, the methyl group is removed from H3K9 by mechanisms such as REDOX reactions. H3K9 is a lysine 9 site on histone H3 that plays a key role in the structural and functional regulation of chromatin. H3K9 can undergo monomethylation (H3K9me1), dimethylation (H3K9me2), and trimethylation (H3K9me3)[ 27 ].These changes in methylation status have varying effects on the gene expression. H3K9me3 is usually associated with gene silencing, which recruits repressor proteins such as heterochromatin protein 1 (HP1) to keep chromatin in a condensed heterochromatin state, thereby preventing gene access by transcriptional factors and others[ 28 , 29 ]. Contrastively, H3K9me1/2 is often associated with gene activation or other more dynamic chromatin regulation processes. Abnormal H3K9 modifications have been associated with a variety of diseases[ 30 , 31 ]. In cancer, the genomes of cancer cells tend to show disturbed H3K9 methylation patterns, leading to activation or silencing of these genes to promote tumorigenesis and progression. KDM3B alters the local state of chromatin primarily by reducing H3K9me1 and H3K9me2 to an unmethylated state, thereby affecting the chromatin structure and gene expression[ 32 ]. We detected the H3K9me1 and H3K9me2 levels in KDM3B knockdown and overexpressed CRC cells by Western blot, finding that the H3K9me2 level changed with the change of KDM3B expression. Thus, in CRC, KDM3B promotes tumorigenesis and progression by modulating the level of H3K9me2. NI refers to the infiltrative growth of tumor cells along the nerve fibers, perineurium, or endoneurium. As a special mode of tumor cell invasion, it can form tiny tumor lesions around the nerves and spread along the nerve direction[ 33 , 34 ]. In CRC, NI is considered an important prognostic indicator for patients, and when cancer cells invade the nerve tissues, the survival rate of patients may be reduced, with a corresponding increase in the risk of recurrence and metastasis[ 35 , 36 ]. When performing surgery or pathological analysis, doctors pay special attention to the presence or absence of NI, in order to more accurately predict the disease development and formulate corresponding therapeutic regime. In our study, H&E staining was performed on the tumors from CRC NI model mice, finding that tumors overexpressing KDM3B had evidently severer invasion of sciatic nerves than the control group. Suggestively, KDM3B exerts a role in inducing CRC NI, while inhibiting the KDM3B expression can effectively improve the CRC prognosis. When NI occurs in tumors, there is also a bidirectional interaction between the nervous system and tumors, and the regulatory mechanism of nervous system in tumor tissues is gradually revealed[ 37 – 39 ]. By secreting many cytokines, the central and peripheral nerves are involved in tumorigenesis, growth and metastasis[ 40 , 41 ], which also exert indirect effects on the tumor immunity and microenvironment[ 42 ]. In our in vitro study of co-culture between CRC cells and mouse DRGs, we observed that the DRGs co-cultured with KDM3B-overexpressing CRC cells grew more rapidly than the control group, which enabled them to secret many cytokines, thereby activating the downstream pathways after binding to neurotrophic receptor to drive the CRC progression. Through a combination of mRNA-Seq, Cut-tag and ATAC-Seq analyses, we found that KDM3B activated the NTRK1 initiation sequence by upregulating H3K9me2, thus causing the overexpression of neurotrophic receptor TrkA and its binding with NGF to activate the MAPK and PI3K pathways[ 43 , 44 ]. PI3K (phosphatidylinositol 3-kinase) plays a critical role in tumor invasion. It can regulate the cell motility and endow tumor cells with stronger migration ability by influencing the cytoskeleton recombination, so that the tumor cells can break natural barriers such as the basement membrane around tumor tissues to infiltrate into the surrounding normal tissues[ 45 , 46 ]. The MAPK (mitogen-activated protein kinase) pathway also plays a critical role in the tumor invasion process, and its abnormal activation can promote the migration and invasion abilities of tumor cells. MAPK degrades the extracellular matrix components by upregulating the matrix metalloproteinases (MMPs) and other related proteins, thereby opening up a channel for the tumor cell invasion to help the tumor cells infiltrate into surrounding tissues and metastasize into distant tissues[ 47 , 48 ]. The MAPK and PI3K pathways profoundly affect the progression of tumors and the prognosis of patients, which have a high potential as targets in tumor therapy research. Inhibiting the KDM3B expression can suppress the activation of MAPK and PI3K pathways from upstream to inhibit the CRC NI, thereby improving the prognosis of CRC patients. The present study explored the mechanism of CRC NI through multi-omics combined with in vivo and in vitro experiments, and conducted in vivo and in vitro validation. The results revealed that KDM3B activated the initiation sequence of NTRK1 by demethylating H3K9me2, leading to the overexpression of neurotrophic receptor TrkA. Binding between TrkA and NGF overactivated the MAPK and PI3K pathways to promote the NI in CRC. Our study provides novel targets for treating CRC patients with NI, as well as new ideas for improving their prognosis. Abbreviations Transposase Accessible Chromatin with high-throughput sequencing (ATAC-Seq) Brain-derived neurotrophic factor (BDNF) Cytosine-phosphate-guanine (CpG) Colorectal cancer (CRC) Cleavage under targets and tagmentation (CUT&Tag) Differentially expressed proteins (DEGs) Dulbecco's modified Eagle medium (DMEM) Dorsal root ganglia (DRGs) Eastern Cooperative Oncology Group (ECOG) Fetal bovine serum (FBS) Heterochromatin protein 1 (HP1) Lysine demethylase 3B (KDM3B) Neural invasion (NI) Nerve growth factor (NGF) Neurotrophic factor 4 (NT-4) Tyrosine kinase receptor A (TrkA) Tyrosine kinase receptor B (TrkB) Tyrosine kinase receptor C (TrkC) Transcription start sites (TSS) Declarations Acknowledgment: We thank all authors who contributed valuable methods and data and made them public. Data Availability Statement: All the other data supporting the findings of this study are available within the article and its Supplementary Information Files, or from the corresponding authors upon reasonable request. Disclosure Statement: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Funding: This work was supported by Social Development Plan of Taizhou, China(Grant No.TS202419) and Suqian Sci & Tech Program (Grant No.KY202212) Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This work described has not been published previously and not under consideration for publication elsewhere. Ethics Statement: All procedures performed in studies involving human participants were in accordance with 1964 Helsinki declaration and its later amendments or comparable ethical standards. The clinical research section of this study was approved by Ethics Committee of Affiliated Taizhou People’ s Hospital of Nanjing Medical University (KY2023-164-01). All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. The animal experimental protocols in this study were approved by the Huachuang Sino Animal Experiment Ethics Committee (SK23035-P001-01). Authors Contribution: G. C. conceived and designed this study. G. C., J. L., W. X. and S. S. Performed experiments and/or analyzed data and/or prepared the figures. G. C., X. J. and X. F. analyzed data. G. C and H. Z. wrote the first version of the manuscript. X. Z., J. L. and Y. L. revised the manuscript. X. F. and J. 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(A) Differential proteins in the control group vs. NI group; (B) Volcano plot of differential proteins; (C) Localization of differential proteins; (D) GO enrichment analysis of differential proteins; (E) KEGG enrichment analysis of differential proteins; (F) GSEA enrichment analysis based on GO enrichment; (G) GSEA enrichment analysis based on KEGG enrichment; (H) Immunohistochemistry of KDM3B proteins in colon cancer tissues of patients.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568594/v1/73f895e67c911a58c5dc09f5.jpg"},{"id":88413912,"identity":"a987c70d-ca37-4ca2-b3ce-cc4af645ec18","added_by":"auto","created_at":"2025-08-06 08:43:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20203822,"visible":true,"origin":"","legend":"\u003cp\u003eKDM3B induces NI in colon cancer cells in the presence of neurokines.(A) Knockdown and overexpression of KDM3B in colon cancer cell lines HCT116 and HT29; (B) Detection of migration level of colon cancer cell lines after knockdown and overexpression of KDM3B; (C) Detection of invasion level of colon cancer cell lines after knockdown and overexpression of KDM3B; (D) Schematic diagram of colon cancer NI model in vitro; (E) Detection of NI level of colon cancer cell lines after knockdown and overexpression of KDM3B; (F) Detection of migration level of colon cancer cell lines after addition of neurokines; (G) Detection of invasion level of colon cancer cell lines after knockdown and overexpression of KDM3B after addition of neurokines.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568594/v1/0ed169442c461341bfc8246b.jpg"},{"id":88415126,"identity":"46b8ed96-a9e3-484d-99af-a2c404e62cec","added_by":"auto","created_at":"2025-08-06 08:51:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7679384,"visible":true,"origin":"","legend":"\u003cp\u003eNTRK1 interact with KDM3B derived by multi-omics.(A) mRNA-Seq DRGs (sh-NC vs. sh-KDM3B); (B) GO and KEGG enrichment analysis based on the mRNA-Seq DRGs; (C) Heatmap of DRGs binding to KDM3B detected by Cut-tag (sh- NC vs. sh-KDM3B); (D) Cut-tag binding DRGs and functional categorization; (E) GO and KEGG enrichment analysis based on Cut-tag DRGs; (F) Heatmap of differences in chromatin open levels of sequences bound to KDM3B detected by ATAC-Seq (sh-NC vs. sh-KDM3B); (G) Heatmap of ATAC- Seq DRGs for functional classification and TSS distances; (H) GO and KEGG enrichment analysis based on ATAC-Seq DRGs; (I) Wayne plots of multilocus sequencing; (J) NTRK1 in multi-omics; (K-N) Differences in serum levels of NGF, BDNF, NT-3, and NT-3 in the patients; (O) CRC tissues of the patients for TrkA, TrkB, TrkC protein immunohistochemistry.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568594/v1/4b51aa1032fbc7fc0acdbf97.jpg"},{"id":88413910,"identity":"1606fb92-094d-4a86-8330-840e2f9d830f","added_by":"auto","created_at":"2025-08-06 08:43:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4948691,"visible":true,"origin":"","legend":"\u003cp\u003eKDM3B induces CRC NI by inhibiting H3K9me2 from Cut-tag. (A) Schematic diagram of KDM3B action sites; (B) Heatmap of DRGs binding to H3K9me1 as detected by Cut-tag (sh-NC vs. sh-KDM3B); (C) Functional classification of the Cut-tag (H3K9me1) DRGs with functional classification and TSS distances; (D) GO and KEGG enrichment analysis based on Cut-tag (H3K9me1) DRGs; (E) Heatmap of DRGs bound to H3K9me2 detected by Cut-tag (sh-NC vs. sh-KDM3B); (F) Cut-tag (H3K9me2) DRGs with functional classification and TSS distances; (G) GO and KEGG enrichment analysis based on Cut-tag (H3K9me2) DRGs; (H) NTRK1 in Cut-tag (H3K9me1) and Cut-tag (H3K9me2); (I) Effect of KDM3B levels on H3K9me1 and H3K9me2.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568594/v1/e987dc87d80b031aa446998f.jpg"},{"id":88415136,"identity":"75726664-ac52-431c-8189-abbfdad6e698","added_by":"auto","created_at":"2025-08-06 08:51:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14178384,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of NTRK1 reverses KDM3B-induced NI in CRC. (A) Knockdown and overexpression of NTRK1 by CRC cell lines HCT116 and HT29; (B) Knockdown of NTRK1 reverses the promotional effect of KDM3B on the migration level of CRC cell lines; (C) Knockdown of NTRK1 reverses the promotional effect of KDM3B on the invasion level of CRC cell lines; (D) Knockdown of NTRK1 reverses the promotional effect of KDM3B on the promotion of NI level.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568594/v1/e793fffc96ed23b1a4f0521b.jpg"},{"id":88413921,"identity":"666dc1d4-14bf-4d04-b92d-ba1a57debeef","added_by":"auto","created_at":"2025-08-06 08:43:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13628244,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo model validation of KDM3B-promoted CRC NI. (A-C) Differences in tumor size in CRC NI model (Control vs. sh-KDM3B); (D) Differences in NI levels in CRC NI model (Control vs. sh-KDM3B); (E) Differences in TrkA expression levels in CRC NI model (Control vs. sh-KDM3B); (F) CRC cell lines, the effect of KDM3B expression level on MAPK pathway and PI3K pathway; (G) Differences in the activation level of MAPK pathway and PI3K pathway in CRC NI models (Control vs. sh-KDM3B).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568594/v1/4ad3e386173898a25c369699.jpg"},{"id":88417172,"identity":"40aad971-b09b-4e32-93c9-2bd500047e19","added_by":"auto","created_at":"2025-08-06 09:08:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":48513138,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6568594/v1/c66a8d88-7943-41c7-8a30-5deeb5f7ac4f.pdf"},{"id":88413909,"identity":"2dc86907-a64c-4c9a-ab6c-f20b66eaa1ac","added_by":"auto","created_at":"2025-08-06 08:43:29","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1215065,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6568594/v1/424b004a5defaa11e743f11e.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eKDM3B promotes neural invasion in colorectal cancer through TrkA upregulation by inhibiting H3K9 dimethylation\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC), as the third most common cancer in the world with a mortality rate ranking the third, poses a serious threat to human health[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Metastasis is a key risk factor for poor prognosis in patients with CRC. The 5-year survival rate of metastatic CRC is less than 15% [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and neural invasion (NI) is a major risk factor for local recurrence and distant metastasis of CRC[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Systemic and local cross-linking between the nervous system and tumors has become a new hotspot in the tumor control research[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In CRC, NI can drive the tumor neovascularization and induce the tumor metastasis[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Besides, nervous system disorders can also affect the immune system and promote the neoplastic tumorigenesis[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Understanding the mechanism of NI in CRC and inhibiting its occurrence can help improve the survival rate and quality of life of patients.\u003c/p\u003e\u003cp\u003eNeurotrophic receptors are expressed on the surface of various tumor cells, with common ones including tyrosine kinase receptor A (TrkA), tyrosine kinase receptor B (TrkB), and tyrosine kinase receptor C (TrkC) encoded by the NTRK gene. Nerve growth factor (NGF) binds to TrkA[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], brain-derived neurotrophic factor (BDNF) and neurotrophic factor 4 (NT-4) both bind to TrkB [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and neurotrophic factor 3 (NT-3) binds to TrkC[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Different neurotrophic receptors tend to exhibit different physiological processes after binding to neurotrophic factors. In tumors, there are abnormalities in NTRK gene expression[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], which causes the overexpression of neurotrophic receptors on the tumor cell surface. Meanwhile, excessive binding of neurotrophic receptors to neurotrophic factors can activate the MAPK, PI3K and PLCγ pathways in tumors[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], thereby inducing the malignant tumor progression, which is especially critical in the NI process [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Hence, reducing the binding between neurotrophic receptors and factors becomes crucial to mitigate the NI in CRC.\u003c/p\u003e\u003cp\u003eDNA methylation, as an important mode of epigenetic modification, occurs predominantly on cytosine in cytosine-phosphate-guanine (CpG) dinucleotides[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Abnormal DNA methylation plays a pivotal role in tumor biology, which causes the silencing of some tumor suppressor genes, the activation of proto-oncogenes, and the abnormal expression of transposons, increasing genomic instability and creating favorable conditions for tumorigenesis and development[\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The in-depth study of tumor methylation not only facilitates better understanding of the tumor pathogenesis, but also provides new ideas and directions for early diagnosis, prognostic assessment and targeted therapy of tumors.\u003c/p\u003e\u003cp\u003eIn this study, through proteomic sequencing on the clinical tissues of CRC patients with or without NI, we detected the highly expressed protein KDM3B in neuroinvasive CRC tissues. As a histone demethylase, lysine demethylase 3B (KDM3B) often acts on H3K9me1 and H3K9me2[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The downstream sequence NTRK1 of KDM3B action on H3K9me1 and H3K9me2, as well as its chromatin openness in CRC cells before and after KDM3B knockdown were detected by CHIP-Seq and ATAC-Seq.\u0026nbsp;TrkA was identified as the downstream site of KDM3B's role in CRC, which was then verified through in vivo and in vitro experiments. The present study reveals the mechanism whereby KDM3B promotes NI in CRC by the H3K9me2 demethylation-mediated upregulation of TrkA, laying a solid theoretical foundation for the application of KDM3B as a prognostic indicator and therapeutic target for CRC (Graphical abstract).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eSubject inclusion and exclusion criteria\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe clinical part of this study was approved by the Ethics Committee of The Affiliated Taizhou People's Hospital of Nanjing Medical University (KY2023-164-01). Surgically resected tumor tissues from six CRC patients, three without NI and three with NI, were collected for the follow-up study. Inclusion criteria: 1. CRC confirmed by colonoscopy and pathological biopsy; 2. Patients aged between 18 and 75 years; 3. Newly diagnosed patients who\u0026nbsp;have not yet received any anticancer therapy; 4. Eastern Cooperative Oncology Group (ECOG) score of 0-2 points; 5. After being fully informed of the research purpose, method, process, potential risks and benefits, patients or their legal representatives voluntarily signed informed consent and agreed to participate in this clinical study. Exclusion criteria: 1. Complicated with cardiovascular and cerebrovascular diseases, severe hepatic and renal insufficiency, other serious diseases such as malignant tumors; 2. Mental or cognitive impairment; 3. Patients with poor compliance; 4. Received a specific treatment or trial within 3 months.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCell culture and transfection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe STR-validated ovarian cancer cell lines HCT116 and HT29, which were procured from Procell Life Sciences (China), were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). To silence lysine demethylase 3B (KDM3B), we transfected the HCT116 and HT29 cells with lentivirus sh-KDM3B and sh-NTRK1 plasmids that were chemically synthesized by GenePharma (Shanghai, China). The sequences of sh-KDM3B were as follows: sh-KDM3B#1: 5'-CCGATGTGAAAGTTCGGGATT-3'; sh-KDM3B#2: 5'-GCGATCTTTGTAGAATTTGAT-3'; sh-KDM3B#3: 5'-GTTAGTCTTCCTTTCGATAAA-3'. The sequences of sh-NTRK1 were as follows: sh-NTRK1#1: 5'-TATCTACAGCACCGACTATTA-3'; sh-NTRK1#2: 5'-TGCCTTCATGGACAACCCTTT-3'; sh-NTRK1#3: 5'-CATCGAGAACCCACAATACTT-3'.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCell migration and invasion assays\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the migration ability of cells, we created a linear wound on each nearly fused monolayer of HCT116 and HT29 cells. Images were acquired at 0, 24, and 48 h after scratching to measure the scratch closure for quantifying cell migration. The invasion ability of the cells was detected by Transwell assay. The Matrigel-coated upper chamber of Transwell inserts was added with 1×10\u003csup\u003e4\u003c/sup\u003e HCT116 and HT29 cells, while the lower chamber was supplemented with a culture medium containing 20% FBS. Three d later, non-migrated cells were removed, and the migrated cells were fixed, stained, counted, and photographed under an inverted microscope (Nikon, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDRG tumor-cell co-culture assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of 2×10\u003csup\u003e5\u003c/sup\u003e CRC cell lines, HCT116 and HT29, were suspended in 25 µL of growth factor-reduced Matrigel (Sigma-Aldrich, USA) and placed in the center of each of the 12 wells. The dorsal root ganglia (DRGs) of each 8-d-old Sprague-Dawley (SD) rats were extracted, and inoculated with 25 µL of Matrigel near the cell suspension, while an additional 25 µL of blank Matrigel was added on the opposite side (Figure 2.D) [23]. The Petri dishes were incubated at 37°C in CO\u003csub\u003e2\u003c/sub\u003e with 5% humidity for 30 min to allow solidification. Thereafter, the cultures were incubated with 10% FBS-containing NeuroBasal medium (Invitrogen, USA). The degree of tumor NI was observed microscopically at d 0, 3, and 7, and images were collected.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWestern Blot Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTotal proteins were extracted from HCT116 and HT29 cells using RIPA lysis buffer containing phenylmethylsulfonyl fluoride (Beyotime, USA). After quantification with BCA protein assay kit, equal amounts of proteins were separated by 10% SDS-PAGE and then transferred onto PVDF membranes. Subsequently, the membranes were blocked in 5% skim milk powder at room temperature for 2 h, and then incubated with primary antibodies against KDM3B (MA5-32123; Thermo Fisher Scientific, USA) and TrkA (PA5-17170; Thermo Fisher Scientific, USA) overnight at 4°C. Finally, the membranes were incubated with HRP-conjugated secondary antibody at room temperature for 2 h, followed by the observation of chemiluminescent signals using Pierce™ ECL substrates (Thermo Fisher Scientific, USA).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003emRNA sequencing (mRNA-Seq)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003emRNA-Seq was accomplished by LC-Bio Technologies (Hangzhou) Co., Ltd. The cells were lysed in Trizol (Takara, Japan), total RNA was prepared using RNA Clean \u0026amp; Concentrator-5 ™ (Zymo, USA), and genomic DNA contamination was eliminated using DNase. After isolating with the NEBNext PolyA mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, USA), the mRNA was used for RNA-Seq library preparation by the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, USA). The libraries were sequenced in Illumina with the paired-end 2 x 150 sequencing mode.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAssay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-Seq)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of 50,000 viable cells were collected, washed in cold PBS and centrifuged at 500 g and 4°C for 5 min. Each sample was resuspended with 50 μl of lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 0.1 % IGEPAL, 0.1 % NP-40, 0.1 % Tween-20) and then incubated on ice for 10 min. The supernatant was discarded after 5 min of centrifugation at 500 g and 4 °C, while the cell nuclei were collected for subsequent transposition reaction. Library construction was performed following the instructions of TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, China). Finally, the libraries were sequenced on the Illumina Novaseq 6000 platform at 150 bp paired-end read lengths by Genefund Biotech (Shanghai).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCleavage under targets and tagmentation (CUT\u0026amp;Tag)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCUT\u0026amp;Tag assays were conducted on fresh WDLPS, DDLPS, and PA cells (100,000 cells/sample), treated with or without 0.8 μM QN-302, for 4 h. After collection and washing with wash buffer, the cells were immobilized on concanavalin A-coated magnetic beads (Bangs Laboratories Inc., USA) and incubated overnight at 4°C with 500 ng FLAG-tagged anti-G4 BG4 antibody. Then, the samples were incubated at RT for 1 h with anti-FLAG antibody (Merck, USA), and further with rabbit anti-mouse IgG (Merck, USA). Bead-bound cells were washed in dig-wash buffer, bound with pA–Tn5 adapter complex, and tagmented at 37°C for 1 h. After the tagmentation, libraries were purified by phenol–chloroform extraction and amplified using NEBNext High-Fidelity 2× PCR Master Mix with barcoded i5 and i7 primers. CUT\u0026amp;Tag libraries were size-selected with Agencourt AMPure XP Beads (Beckman Coulter, USA) and size-analyzed by Bioanalyzer (Agilent Technologies, Italy). Finally, the samples were sequenced on an Illumina NextSeq500 platform with paired-end 38 bp reads.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo NI model\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe in vivo NI model was established by a modified method based on a previously described protocol [24]. Four-weeks-old male nude mice were anesthetized by methoxyflurane inhalation for subsequent surgery. The sciatic nerve was surgically exposed and 5 μL of 2×10\u003csup\u003e5\u003c/sup\u003e/μL CRC cell suspension was injected around it. To assess the effect of KDM3B knockdown on NI, the tumor volume as well as sciatic nerve-related hindlimb behavior were examined every 7 d. Four weeks later, the mice were euthanized and the xenografts were excised to compare tumor size. This work has been reported in accordance with the ARRIVE guidelines[25].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData from three independent experiments were expressed as means ± standard deviations. Differences between two groups were analyzed by paired or unpaired Student's t-test, while differences among multiple groups were evaluated by one-way ANOVA and Tukey's test. The correlation between gene expression data was tested with Pearson's correlation coefficient. P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eProteomic screening of KDM3B, a key protein inducing NI in CRC\u003c/h2\u003e\u003cp\u003eTo explore the key proteins responsible for inducing NI in CRC, we clinically collected surgical tumor tissues from six patients with pathological stage III CRC complicated by vascular invasion, including three patients in the NI group and three patients in the non-NI group. The tumor tissues from the two groups were subjected to differential protein analysis by TMT-Seq.\u0026nbsp;Compared with the NI group, there were 98 differentially expressed proteins (DEGs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A-B) in the non-NI group, with 27 DEGs located in the cell nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.C), accounting for the majority, among which the KDM3B protein was the most differentially expressed. As a histone demethylase, KDM3B plays an important role in biological processes such as gene expression regulation, and affects the expression of multiple proteins by changing the methylation level of histones.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese DEGs were subjected to GO and KEGG enrichment analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.D-E) for investigating their effects on the CRC functions. GSEA enrichment analyses were separately performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.F-G) on the basis of GO and KEGG enrichment. The results showed that the DEGs could be enriched into \"GO:0016477 cell migration\", \"GO:0030335 positive regulation of cell migration\", \"hsa04010 MAPK signaling pathway\", and \"hsa04151 PI3K-AKT signaling pathway\" that are associated with tumor migration and invasion.\u003c/p\u003e\u003cp\u003eThe KDM3B levels of the patients were validated by immunohistochemistry. According to our results, the three patients in the NI group all exhibited higher KDM3B levels than the three non-NI patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.H). Suggestively, KDM3B plays a crucial role in the NI process of CRC.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eBinding between neurotrophic receptors and factors was required during NI in CRC\u003c/h2\u003e\u003cp\u003eTo verify whether KDM3B was associated with the migration and invasion of CRC, we first conducted in vitro experiment by knocking down and overexpressing KDM3B in CRC cell lines HCT116 and HT29 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A). For the subsequent experiment, sh-KDM3B#2 with the highest knockdown efficiency and the overexpressed oe-KDM3B were selected. Scratch and Transwell assays were applied to examine the cell migration and invasion abilities. In the normal culture environment, the KDM3B level had insignificant effects on the migration and invasion abilities of CRC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.B-C), which was inconsistent with our proteomics results.\u003c/p\u003e\u003cp\u003eTo further investigate the interaction between CRC cells and nerves, we established a co-culture model of SD rat DRGs and CRC cells that was closer to the in vivo environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.D). After 5 d of co-culture between DRGs and CRC cells, the tumor cells began to migrate towards the DRGs, and the DRG axons started to grow towards the CRC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.E). After 7 d of co-culture, we began to compare the migration distance of tumor cells and the growth level of DRGs among the groups. The results showed that the migration distances of HCT116 and HT29 cells were significantly shorter in the sh-KDM3B group than in the control group, while the oe-KDM3B group exhibited significantly longer migration distances than the control group. Comparison of the DRG growth level found that the coverage of DRGs in the sh-KDM3B group was significantly smaller than that of the control group, while the oe-KDM3B group displayed significantly larger coverage than the control group. Suggestively, the DRG growth level was poor in the sh-KDM3B group, whereas was good in the sh-KDM3B group.\u003c/p\u003e\u003cp\u003eExcessive binding between the neurotrophic receptors and factors can induce NI in tumors. Changes in both the neurotrophic receptor and factor expressions can affect the degree of tumor NI. By comparing the differences in growth environment between the above experimental tumor cells, we conjectured that the binding between neurotrophic receptors and factors might be involved in the NI process of CRC in synergy with KDM3B. By adding the neurotrophic factors on the basis of the previous experimental conditions, we reexamined the migration and invasion abilities of CRC cells through scratch and Transwell assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.F-G). It was found that under the synergy of neurotrophic factors, the migration and invasion abilities were significantly weaker in the sh-KDM3B group than in the control group, while the oe-KDM3B group exhibited significantly stronger migration and invasion abilities than the control group. Thus, KDM3B requires the binding of neurotrophic receptors to neurotrophic factors in the process of inducing CRC NI.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eKDM3B induced CRC NI by triggering TrkA overexpression\u003c/h2\u003e\u003cp\u003eTo investigate the specific reasons for the elevated degree of neurotrophic receptor and factor binding in CRC patients with NI, we first performed transcriptomic sequencing on the HCT116 and HT29 CRC cell lines treated with sh-vector and sh-KDM3B, finding a total of 2,021 common DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A). These DEGs were subjected to the GO and KEGG enrichment analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.B), and the results showed that they could be enriched into such pathways as \"GO:0016477 RNA splicing\", \"GO:0030335 nuclear transport\", and \"GO:0030335 protein localization to nuclear body\", which are associated with the nuclear entry and role of proteins. In view of the entry and role of KDM3B in the cell nuclei, we next explored the differences in KDM3B-binding sequences by performing Cleavage Under Targets and Tagmentation (Cut-tag) on the sh-NC and sh-KDM3B-treated CRC cell lines HCT116 and HT29 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.C). A total of 2,860 inter-group differentially expressed KDM3B-binding DNA sequences were identified, most of which were promoter sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.D). Through the GO and KEGG enrichment analyses of the inter-group differentially expressed sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.E), we found that the DEGs could be enriched into such pathways as \"GO:0001841 neural tube formation\". \"GO:0002467 germinal center formation\", and \"hsa:04390 Hippo signaling pathway\", which are associated with the tumor NI. We further examined the chromatin accessibility of sh-vector and sh-KDM3B-treated HCT116 and HT29 cells by ATAC-Seq assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.F). A total of 2,749 inter-group differentially expressed sequences of chromatin openness were identified, most of which were promoter sequences localized near the transcription start sites (TSS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.G). According to the GO and KEGG enrichment analyses of inter-group DEGs obtained after the ATAC-Seq analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.H), the DEGs could be enriched into such pathways as \"GO:0010975 regulation of neuron projection development\", \"hsa:04015 Rap1 signaling pathway\", and \"hsa:04520 Adherens junction\", which are related to nerve growth. Thus, KDM3B is somewhat associated with NI in CRC cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe intersected the DEGs obtained from mRNA-Seq, Cut-tag and ATAC-Seq analyses to identify a total of 155 common DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.H). Among them, TrkA-encoding NTRK1 was significantly differentially expressed in all of the mRNA-Seq, Cut-tag and ATAC-Seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.J). Therefore, the level of NTRK1 involved in transcription was downregulated after KDM3B knockdown, and at the same time, the chromatin of NTRK1 was more difficult to access. Knockdown of KDM3B could inhibit the transcriptional efficiency of NTRK1. NTRK genes encode the neurotrophic receptors TrkA, TrkB, and TrkC, with NTRK1 being the TrkA-encoding gene. Overactivation of NTRK1 could increase the TrkA expression level and induce CRC NI after binding with NGF. Thus, in CRC, KDM3B triggers overexpression of TrkA to induce NI.\u003c/p\u003e\u003cp\u003eFurther, the above sequencing results were verified by detecting the expression levels of neurotrophic factors and receptors in clinical patients. We collected sera from 60 pathological stage III CRC patients with vascular invasion, including 30 patients in the NI group and 30 patients in the non-NI group. Elisa was applied to examine the differences in the serum levels of NGF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.K), BDNF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.L), NT-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.M) and NT-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.N) between the two groups. The results suggested no significant inter-group differences in the NGF, BDNF, NT-3 or NT-3 expression. Immunohistochemistry was performed to assess the differences in the neurotrophic factor expression in the tumor tissues of patients in the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.O), finding that the TrkA level in the NI group was significantly higher than that in the non-NI group, thus verifying our sequencing results.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eKDM3B triggered TrkA overexpression by inhibiting H3K9me2\u003c/h2\u003e\u003cp\u003eKDM3B, as a histone demethylase, tends to act on H3K9me1 and H3K9me2, thereby affecting the expression of downstream genes and proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.A). We applied Western blot to detect the expression levels of H3K9me1 and H3K9me2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.B), finding that the H3K9me2 level in the sh-KDM3B group was significantly lower than that in the control group, while the oe-KDM3B group exhibited significantly higher H3K9me2 level than the control group. No significant inter-group differences were noted in the H3K9me1 level.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further explore the specific mechanism whereby KDM3B promotes the TrkA expression in CRC, we applied Cut-tag to assess the differences in H3K9me1- and H3K9me2-binding sequences in CRC cells before and after the KDM3B knockdown. The results showed that after knocking down KDM3B, there were differences in H3K9me1-binding sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.C), and the inter-group differentially expressed sequences were mostly in the promoter region and localized near the TSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.D). The DEGs could be enriched into pathways like \"GO:0007059 chromosome segregation\", \"GO:0006260 DNA replication\", and \"hsa:04115 p53 signaling pathway\" that are closely associated with tumor progression, but failed to be enriched into the tumor NI-related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.E). Differences in H3K9me2-binding sequences were more significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.F), with more number of promoter sequences and denser localization near the TSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.G). The DEGs could be enriched into such pathways as \"GO:0071897 DNA biosynthetic process\", \"GO:0006275 regulation of DNA replication\", and \"hsa:04152 AMPK signaling pathway\", which are associated with abnormal biological behaviors of tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.H). By analyzing the NTRK1 level of H3K9me1- and H3K9me2-binding sequences, we found that the NTRK1 level of H3K9me2-binding sequences changed significantly before and after the KDM3B knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.I). Thus, in CRC, KDM3B is more likely to trigger TrkA overexpression via H3K9me2, ultimately inducing NI.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eNTRK1 knockdown could reverse the KDM3B-induced NI\u003c/h2\u003e\u003cp\u003eTo further verify our sequencing results, we validated the NI-related phenotypic responses in CRC HCT116 and HT29 cells.\u003c/p\u003e\u003cp\u003eInitially, we knocked down and overexpressed TrkA in the CRC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.A), and selected sh-NTRK1#1 with the highest knockdown efficiency and the overexpressed oe-NTRK1 for the subsequent experiment. By observing changes in the cell migration and invasion abilities, the NTRK1 level was found to have no significant effect on the migration and invasion abilities of CRC cells in the normal culture environment. After adding neurotrophic factors, the migration and invasion abilities were significantly weaker in the sh-NTRK1 group than in the control group, while the oe-NTRK1 group exhibited significantly stronger migration and invasion abilities than the control group. Overexpression of NTRK1 after KDM3B inhibition partially restored the declined migration and invasion abilities (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.B-C), demonstrating that KDM3B could enhance the migration and invasion abilities of CRC cells by upregulating NTRK1. In the co-culture model of DRGs and CRC cells, the migration distance of tumor cells was significantly shorter in the sh-NTRK1 group than in the control group, while the oe-NTRK1 group displayed significantly longer migration distance than the control group. Overexpression of NTRK1 after KDM3B inhibition could partially restore the reduced neural migration distance. Meanwhile, the coverage of DRGs in the sh-NTRK1 group was significantly smaller than that in the control group, while the oe-NTRK1 group exhibited significantly larger coverage than the control group. Overexpression of NTRK1 after KDM3B inhibition could restore the narrowed nerve coverage. This suggests that NTRK1 can directly affect the degree of NI and the growth level of DRGs in CRC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe above experiments demonstrated that inhibition of NTRK1 could reverse the NI induction by KDM3B in CRC, thus further verifying that KDM3B can induce CRC NI by triggering the overexpression of TrkA-encoding NTRK1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eValidation of in vivo model of CRC NI promoted by KDM3B\u003c/h2\u003e\u003cp\u003eTo further verify our in vitro results, a mouse model of CRC NI was established for in vivo experimental validation. Normal and KDM3B knockdown CRC cells were injected separately around the right and left sciatic nerves of nude mice, during which the bilateral tumor sizes were measured. Four weeks later, the mice were sacrificed, and the degree of bilateral tumor NI was observed by H\u0026amp;E staining. The results suggested that in the nude mouse NI model established by HCT116 and HT29, the right side KDM3B knockdown tumors were significantly smaller than the left side normal tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.A-C), thus proving that KDM3B can promote the growth of CRC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the effect of KDM3B on the degree of NI in CRC, H\u0026amp;E staining was performed on bilateral tumors to observe the NI degree. The results showed that in the nude mouse NI model established by HCT116 and HT29, the degree of NI in the right side KDM3B knockdown tumors was significantly lower than that in the left side normal tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.D). Meanwhile, we also applied immunohistochemistry to detect the TrkA expression in bilateral tumors, finding that the TrkA level in the right side KDM3B knockdown tumors was significantly lower than that in the left side normal tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.E).\u003c/p\u003e\u003cp\u003eFurther, we validated the enriched pathways and found that in the cellular experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.F) and animal experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.G), the overexpression of KDM3B could activate the CRC MAPK and PI3K pathways, thereby inducing NI in CRC.\u003c/p\u003e\u003cp\u003eTaken together, both in vivo and in vitro experiments demonstrated that KDM3B could induce CRC NI through upregulation of TrkA-encoding NTRK1 by inhibiting H3K9me2.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs a common demethylase, KDM3B plays a crucial role in multiple biological processes, including the regulation of gene expression[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In tumors, KDM3B often plays a role in the malignant process by regulating the methylation level of histone H3K9[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In our study, proteomics was applied to identify KDM3B, the key inducer protein of NI in CRC. Further, the mechanism for KDM3B-induced CRC NI was explored by CHIP-Seq.\u0026nbsp;It was found that KDM3B promoted the expression of neurotrophic receptor TrkA by inhibiting H3K9me2, thereby promoting the NI in CRC (Graphical abstract). Suggestively, KDM3B can be a potential therapeutic target for CRC NI, which helps improve the prognosis of CRC patients.\u003c/p\u003e\u003cp\u003eKDM3B is recognized and bound to nucleosomes containing methylated H3K9 through its specific enzymatically active domain[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Then, utilizing its catalytically active center, the methyl group is removed from H3K9 by mechanisms such as REDOX reactions. H3K9 is a lysine 9 site on histone H3 that plays a key role in the structural and functional regulation of chromatin. H3K9 can undergo monomethylation (H3K9me1), dimethylation (H3K9me2), and trimethylation (H3K9me3)[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].These changes in methylation status have varying effects on the gene expression. H3K9me3 is usually associated with gene silencing, which recruits repressor proteins such as heterochromatin protein 1 (HP1) to keep chromatin in a condensed heterochromatin state, thereby preventing gene access by transcriptional factors and others[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Contrastively, H3K9me1/2 is often associated with gene activation or other more dynamic chromatin regulation processes. Abnormal H3K9 modifications have been associated with a variety of diseases[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In cancer, the genomes of cancer cells tend to show disturbed H3K9 methylation patterns, leading to activation or silencing of these genes to promote tumorigenesis and progression. KDM3B alters the local state of chromatin primarily by reducing H3K9me1 and H3K9me2 to an unmethylated state, thereby affecting the chromatin structure and gene expression[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We detected the H3K9me1 and H3K9me2 levels in KDM3B knockdown and overexpressed CRC cells by Western blot, finding that the H3K9me2 level changed with the change of KDM3B expression. Thus, in CRC, KDM3B promotes tumorigenesis and progression by modulating the level of H3K9me2.\u003c/p\u003e\u003cp\u003eNI refers to the infiltrative growth of tumor cells along the nerve fibers, perineurium, or endoneurium. As a special mode of tumor cell invasion, it can form tiny tumor lesions around the nerves and spread along the nerve direction[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In CRC, NI is considered an important prognostic indicator for patients, and when cancer cells invade the nerve tissues, the survival rate of patients may be reduced, with a corresponding increase in the risk of recurrence and metastasis[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. When performing surgery or pathological analysis, doctors pay special attention to the presence or absence of NI, in order to more accurately predict the disease development and formulate corresponding therapeutic regime. In our study, H\u0026amp;E staining was performed on the tumors from CRC NI model mice, finding that tumors overexpressing KDM3B had evidently severer invasion of sciatic nerves than the control group. Suggestively, KDM3B exerts a role in inducing CRC NI, while inhibiting the KDM3B expression can effectively improve the CRC prognosis.\u003c/p\u003e\u003cp\u003eWhen NI occurs in tumors, there is also a bidirectional interaction between the nervous system and tumors, and the regulatory mechanism of nervous system in tumor tissues is gradually revealed[\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. By secreting many cytokines, the central and peripheral nerves are involved in tumorigenesis, growth and metastasis[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], which also exert indirect effects on the tumor immunity and microenvironment[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In our in vitro study of co-culture between CRC cells and mouse DRGs, we observed that the DRGs co-cultured with KDM3B-overexpressing CRC cells grew more rapidly than the control group, which enabled them to secret many cytokines, thereby activating the downstream pathways after binding to neurotrophic receptor to drive the CRC progression.\u003c/p\u003e\u003cp\u003eThrough a combination of mRNA-Seq, Cut-tag and ATAC-Seq analyses, we found that KDM3B activated the NTRK1 initiation sequence by upregulating H3K9me2, thus causing the overexpression of neurotrophic receptor TrkA and its binding with NGF to activate the MAPK and PI3K pathways[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. PI3K (phosphatidylinositol 3-kinase) plays a critical role in tumor invasion. It can regulate the cell motility and endow tumor cells with stronger migration ability by influencing the cytoskeleton recombination, so that the tumor cells can break natural barriers such as the basement membrane around tumor tissues to infiltrate into the surrounding normal tissues[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The MAPK (mitogen-activated protein kinase) pathway also plays a critical role in the tumor invasion process, and its abnormal activation can promote the migration and invasion abilities of tumor cells. MAPK degrades the extracellular matrix components by upregulating the matrix metalloproteinases (MMPs) and other related proteins, thereby opening up a channel for the tumor cell invasion to help the tumor cells infiltrate into surrounding tissues and metastasize into distant tissues[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The MAPK and PI3K pathways profoundly affect the progression of tumors and the prognosis of patients, which have a high potential as targets in tumor therapy research. Inhibiting the KDM3B expression can suppress the activation of MAPK and PI3K pathways from upstream to inhibit the CRC NI, thereby improving the prognosis of CRC patients.\u003c/p\u003e\u003cp\u003eThe present study explored the mechanism of CRC NI through multi-omics combined with in vivo and in vitro experiments, and conducted in vivo and in vitro validation. The results revealed that KDM3B activated the initiation sequence of NTRK1 by demethylating H3K9me2, leading to the overexpression of neurotrophic receptor TrkA. Binding between TrkA and NGF overactivated the MAPK and PI3K pathways to promote the NI in CRC. Our study provides novel targets for treating CRC patients with NI, as well as new ideas for improving their prognosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTransposase Accessible Chromatin with high-throughput sequencing (ATAC-Seq)\u003c/p\u003e\n\u003cp\u003eBrain-derived neurotrophic factor (BDNF)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCytosine-phosphate-guanine (CpG)\u003c/p\u003e\n\u003cp\u003eColorectal cancer (CRC)\u003c/p\u003e\n\u003cp\u003eCleavage under targets and tagmentation (CUT\u0026amp;Tag)\u003c/p\u003e\n\u003cp\u003eDifferentially expressed proteins (DEGs)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDulbecco\u0026apos;s modified Eagle medium (DMEM)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDorsal root ganglia (DRGs)\u003c/p\u003e\n\u003cp\u003eEastern Cooperative Oncology Group (ECOG)\u003c/p\u003e\n\u003cp\u003eFetal bovine serum (FBS)\u003c/p\u003e\n\u003cp\u003eHeterochromatin protein 1 (HP1)\u003c/p\u003e\n\u003cp\u003eLysine demethylase 3B (KDM3B)\u003c/p\u003e\n\u003cp\u003eNeural invasion (NI)\u003c/p\u003e\n\u003cp\u003eNerve growth factor (NGF)\u003c/p\u003e\n\u003cp\u003eNeurotrophic factor 4 (NT-4)\u003c/p\u003e\n\u003cp\u003eTyrosine kinase receptor A (TrkA)\u003c/p\u003e\n\u003cp\u003eTyrosine kinase receptor B (TrkB)\u003c/p\u003e\n\u003cp\u003eTyrosine kinase receptor C (TrkC)\u003c/p\u003e\n\u003cp\u003eTranscription start sites (TSS)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all authors who contributed valuable methods and data and made them public.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the other data supporting the findings of this study are available within the article and its Supplementary Information Files, or from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare the following financial interests/personal relationships which may be considered as potential competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Social Development Plan of Taizhou, China(Grant No.TS202419) and Suqian Sci \u0026amp; Tech Program (Grant No.KY202212)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This work described has not been published previously and not under consideration for publication elsewhere.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were in accordance with 1964 Helsinki declaration and its later amendments or comparable ethical standards. The clinical research section of this study was approved by Ethics Committee of Affiliated Taizhou People’ s Hospital of Nanjing Medical University (KY2023-164-01).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. The animal experimental protocols in this study were approved by the Huachuang Sino Animal Experiment Ethics Committee (SK23035-P001-01).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG. C. conceived and designed this study. G. C., J. L., W. X. and S. S. Performed experiments and/or analyzed data and/or prepared the figures. G. C., X. J. and X. F. analyzed data. G. C and H. Z. wrote the first version of the manuscript. X. Z., J. L. and Y. L. revised the manuscript. X. F. and J. L. funded this research. All authors have read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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Semin Cancer Biol, 2019; 56: 185-95.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-precision-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjprecisiononcology","sideBox":"Learn more about [npj Precision Oncology](http://www.nature.com/npjprecisiononcology/)","snPcode":"41698","submissionUrl":"https://submission.springernature.com/new-submission/41698/3","title":"npj Precision Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Neural invasion (NI), Colorectal cancer (CRC), Lysine demethylase 3B (KDM3B), H3K9 dimethylation (H3K9me2), Tyrosine kinase receptor A (TrkA)","lastPublishedDoi":"10.21203/rs.3.rs-6568594/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6568594/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeural invasion (NI) represents a critical factor contributing to the unfavorable prognosis of patients diagnosed with colorectal cancer (CRC), necessitating an investigation into its underlying mechanisms. This study identifies lysine demethylase 3B (KDM3B) as a pivotal protein associated with NI in CRC, as determined through tandem mass tag sequencing (TMT-Seq) analysis of clinical CRC tissues, which involved a comparative assessment of control and NI samples. Furthermore, Cut-tag and ATAC-Seq experiments conducted on CRC cell lines (comparing control to sh-KDM3B) demonstrated that KDM3B enhances the expression of the nerve growth factor receptor NTRK1, which encodes the TrkA protein in CRC. This finding was corroborated through both in vivo and in vitro experiments. The results indicated that KDM3B overexpression in CRC results in the inhibition of H3K9me2, which subsequently leads to the upregulation of TrkA. This upregulation facilitates the binding of nerve growth factor (NGF) to TrkA, thereby promoting NI in CRC. This study elucidates the intrinsic mechanisms underlying NI in CRC and provides a robust theoretical framework for considering KDM3B as a prognostic indicator and a potential therapeutic target for the disease\u003c/p\u003e","manuscriptTitle":"KDM3B promotes neural invasion in colorectal cancer through TrkA upregulation by inhibiting H3K9 dimethylation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-06 08:43:24","doi":"10.21203/rs.3.rs-6568594/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-11T02:25:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T01:09:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-19T10:45:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-19T07:49:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33297312001696320117003258267170708630","date":"2025-10-01T21:46:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"78647163512555510462538328611772157359","date":"2025-09-29T23:22:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"193622065012467593299568559465302977229","date":"2025-09-29T21:48:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-04T16:49:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-07T04:25:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-02T10:11:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Precision Oncology","date":"2025-05-01T02:21:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-precision-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjprecisiononcology","sideBox":"Learn more about [npj Precision Oncology](http://www.nature.com/npjprecisiononcology/)","snPcode":"41698","submissionUrl":"https://submission.springernature.com/new-submission/41698/3","title":"npj Precision Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"932c72ed-7b39-485e-b38b-2d3b6fd0cbee","owner":[],"postedDate":"August 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":52722171,"name":"Biological sciences/Cancer"},{"id":52722172,"name":"Biological sciences/Cell biology"},{"id":52722173,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":52722174,"name":"Biological sciences/Genetics"},{"id":52722175,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-11-11T02:38:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-06 08:43:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6568594","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6568594","identity":"rs-6568594","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}