KLF5 regulates actin remodeling to enhance the metastasis of nasopharyngeal carcinoma

KLF5 regulates actin remodeling to enhance the metastasis of nasopharyngeal carcinoma | 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 KLF5 regulates actin remodeling to enhance the metastasis of nasopharyngeal carcinoma Denghui Wei, Zhenyu Yang, Yanfu Peng, Yaqin Wang, Panyang Yang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3390645/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Apr, 2024 Read the published version in Oncogene → Version 1 posted 9 You are reading this latest preprint version Abstract Transcription factors (TFs) engage in various cellular essential processes including differentiation, growth and migration. However, the master TF involved in distant metastasis of nasopharyngeal carcinoma (NPC) remains largely unclear. Here we show that KLF5 regulates actin remodeling to enhance NPC metastasis. We analyzed the msVIPER algorithm-generated transcriptional regulatory networks and identified KLF5 as a master TF of metastatic NPC linked to poor clinical outcomes. KLF5 regulates actin remodeling and lamellipodia formation to promote the metastasis of NPC cells in vitro and in vivo. Mechanistically, KLF5 preferentially occupies distal enhancer regions of ACTN4 to activate its transcription, whereby decoding the informative DNA sequences. ACTN4, extensively localized within actin cytoskeleton, facilitates dense and branched actin networks and lamellipodia formation at the cell leading edge, empowering cells to migrate faster. Collectively, our findings reveal that KLF5 controls robust transcription program of ACTN4 to modulate actin remodeling and augment cell motility which enhances NPC metastasis, and provide new potential biomarkers and therapeutic interventions for NPC. Biological sciences/Cancer/Metastasis Biological sciences/Molecular biology/Transcription Biological sciences/Cell biology/Cell migration Biological sciences/Genetics/Gene regulation Biological sciences/Cell biology/Cytoskeleton/Actin Introduction Metastasis, the heterogeneous and systemic disease, compromises the function of distant organs colonized by aggressive cancer cells, causing disruption of homeostasis and eventually death [ 1 ]. Cancer metastasis is a cell motility-based multistep pathological cascade during which cancer cells disseminate from the primary tumor, hijack tumor microenvironment, intravasate and extravasate from vessels, enter and exit dormancy, accommodate to various tissue conditions and colonize distant organs [ 1 , 2 ]. During metastasis, orchestration of actin cytoskeleton dynamics renders cancer cells sensing and responding to physical stimuli to augment cell motility and dissemination [ 3 – 6 ]. Cells precisely reshape by actin remodeling to resist fluid shear stress in bodily circulatory systems, survive and seed metastatic lesions [ 3 , 5 ]. As the basis of metastasis, cell migration encompasses a series of cellular events. After integrating and transmitting cell-intrinsic stimuli and environment-generated cues, metastatic cells initiate the polarization of actin cytoskeletal machinery at the cell front and subsequently form protrusions [ 6 – 9 ]. Protrusions at the leading edge driven by rearranged dense actin networks generate asymmetric or polarized membrane tension to direct cell migration [ 7 , 10 , 11 ]. Intriguingly, actin-rich nascent membrane protrusions extend from areas with low density of membrane-proximal F-actin [ 12 ]. Lamellipodia, a flatter type of protrusion with fan-like architecture, is dominated by Arp2/3-branched actin, attributed to activation of nucleation-promoting factors by Rho-family GTPases [ 13 , 14 ]. Another mechanism of actin-driven migration shows that, the orchestrated retrograde actin flow creates retrograde shear forces, propelling cells to migrate following the environmental topography [ 9 ]. In response to promigratory signals, rapid transcription modulation by transcription factors (TFs) during cell migration facilitates adaption of cells facing changing extracellular milieu [ 15 ]. Human Krüppel-like factors (KLFs) are a family of 17 transcription factors interacting with DNA through conserved triple C2H2 zinc fingers (ZnFs) domain [ 16 ]. KLFs control various key cellular processes including differentiation, inflammation and migration, with exclusive and redundant functions of each KLF [ 16 – 19 ]. In cancers, derailed expression and function of KLFs under divergent contexts implicate cancer pathogenesis, heterogeneity, therapeutic resistance, recurrence and metastasis [ 16 , 20 ]. A recent study revealed the KLF4-mediated exquisite transcription modulation model involving enhancers [ 21 ]. In the reprogramming of mouse embryonic fibroblasts, KLF4 reorganizes the chromatin structure and rewires three-dimensional enhancer loops, leading to adaptive transcriptional changes [ 21 ]. KLFs cross-regulate and co-opt in cancer development and malignant progression, albeit their intricate regulatory networks and effects remain to be fully determined. Nasopharyngeal carcinoma (NPC) is a nasopharynx epithelium originated carcinoma with a high metastatic proclivity and special geographic distribution that in 2020, > 75% new cases occurred in Eastern Asia and South-Eastern Asia, especially in China [ 22 , 23 ]. After first-line induction chemotherapy plus concurrent chemoradiotherapy treatment, approximately 25% patients still developed recurrence or distant metastasis [ 24 ]. For recurrent or metastatic NPC patients receiving first-line treatment, the median progression-free survival of 7.6 months was suboptimal [ 25 ]. However, the master TF involved in distant metastasis of NPC remains elusive. In this study, we discover that KLF5 operates as a master TF and preferentially occupies distal enhancer regions to regulate transcription output in metastatic NPC. We further show that KLF5 transcriptionally activates ACTN4 by binding to its conserved enhancer to facilitate actin remodeling, extending of actin-rich lamellipodia and distant metastasis of NPC. Results KLF5 is a master TF of metastatic NPC To investigate the master TF engaged in distant metastasis of NPC, we first leveraged the Algorithm for the Reconstruction of Accurate Cellular Networks through Adaptive Partitioning (ARACNe-AP) to build transcriptional interactomes [ 26 ] with a multitude of TF-target interplay derived from gene expression profile of curated datasets (Fig. 1 A). Based on the interactomes across 1,639 known or likely TFs [ 27 ], we generated the intricate TFs regulatory networks by Virtual Inference of Protein-activity by Enriched Regulon analysis based on multiple samples (msVIPER) algorithm to assess transcriptional activity of master TFs [ 28 ] and identified 192 candidates for distant metastasis of NPC (Fig. 1 A and Supplementary Table S1 , 2). We delightedly found that three KLFs (KLF5, KLF7 and KLF3) in 192 active TFs presumably promotes NPC metastasis (Supplementary Fig. S1 A). Compared to other KLFs, the expression of KLF5 ranked top in NPC samples and those with distant metastasis (Fig. 1 B, C). Given its highest activity and expression among three active KLFs in metastatic samples of NPC (Supplementary Fig. S1 A-E), we speculated that KLF5 operates as a master TF and promotes distant metastasis of NPC. We next analyzed two public datasets (GSE103611 and GSE13597) and identified KLF5 high and KLF5 low human NPC groups, and KLF5 high group showed a larger proportion of patients with distant metastasis and stage III–IV disease (Fig. 1 D-G). Gene set enrichment analysis (GSEA) of the transcriptome of these two groups revealed a pronounced enrichment of the metastasis relevant gene set in KLF5 high NPC group (Fig. 1 H). These results indicate that NPC patients with higher expression and transcriptional activity of KLF5 are prone to develop distant metastasis and progress to an advanced stage. KLF5 enhances metastatic potential of NPC cells in vitro and in vivo We compared the expression level of KLF5 in immortalized N2-Tert nasopharyngeal mucosa cells and 6 NPC cell lines. The results showed that KLF5 expressed higher in NPC cells and samples than normal control (Fig. 1 I, J and Supplementary Fig. S2 A). Since KLF5 accelerated cell proliferation and malignant progression of pancreatic ductal adenocarcinoma [ 29 ], short hairpin RNA (shRNA)-mediated knockdown (KD) of KLF5 was used to investigate the role of KLF5 in the carcinogenesis of hyperproliferative NPC. Cell viability and colony formation results showed that, knockdown of KLF5 did not alter the proliferation kinetics of NPC cells (Fig. 2 A and Supplementary Fig. S2 B-E). Therefore, we focused on the role of KLF5 in distant metastasis of NPC and the underlying mechanism. Compared with control cells, knockdown of KLF5 triggered an indolent metastatic phenotype that suppressing cell migration and invasion (Fig. 2 B and Supplementary Fig. S2 F). Moreover, depletion of KLF5 significantly increased the time required for wound closure on 2D surfaces (Fig. 2 C and Supplementary Fig. S2 G, H). Accordingly, overexpression of KLF5 potentiated the motility of NPC cells in vitro (Fig. 2 D, E and Supplementary Fig. S2 I, J). We further constructed the lung metastatic model and inguinal lymph node metastatic model wherein KLF5 -KD or control NPC cells were injected into the tail vein or footpad of immunocompromised mice, respectively. Compared to the control, mice intravenously injected with KLF5 -KD NPC cells shouldered less lung metastatic burden showing reduction of overt metastatic lesions (Fig. 2 F). Consistently, knockdown of KLF5 precluded metastatic dissemination of NPC cells from primary tumor in footpad to groin, leading to smaller tumor-draining lymph nodes (Fig. 2 H). Knockdown of KLF5 constrained NPC cell infiltration of normal tissues including skin, muscle and lung in metastatic models (Fig. 2 G, I). Collectively, our data suggests that KLF5 enhances migratory propensity of cells in vitro and metastatic outbreak in vivo. KLF5 regulates actin remodeling and lamellipodia formation Next, we sought to identify the molecular features and pathways determining KLF5-enhanced metastatic potential of NPC. Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) and Reactome pathway enrichment analysis of KLF5 high and KLF5 low human NPCs in different datasets revealed that the actin cytoskeleton reorganization, Rho-family GTPases and cell motility associated pathways are enriched in KLF5 high group (Fig. 3 A, B). We applied GESA analysis to compare the transcriptome of these two groups, and observed a pronounced enrichment of gene sets germane to actin cytoskeleton, wound healing, Rho-family GTPases and EMT in KLF5 high group (Fig. 3 C, D and Supplementary Fig. S3 A-C). Diverse guidance cues converge into asymmetric or polarized traction force generation to direct cell migration, with leading-edge protrusions acting as one pivotal executor [ 4 ]. Most protrusions are driven by the continuous polymerization and depolymerization of branched and linear arrays of actin, of which lamellipodia appears flatter and is dominated by Arp2/3-branched actin [ 4 , 14 , 30 ]. To evaluate whether KLF5 augments cell motility by tuning actin remodeling and lamellipodia formation, we first implemented RNA-sequencing (RNA-seq) analysis of KLF5 -KD and control NPC cells. By KEGG, GO and GSEA analysis on the transcriptome of KLF5 -KD and control cells, we identified enriched functional categories of actin cytoskeleton, focal adhesion, wound healing and cell migration regulation (Fig. 3 E, F and Supplementary Fig. S3 D-F). Second, we conducted knockdown or overexpression of KLF5 in another individual assay to verify the transcriptomic changes occurring in KLF5 -depleted metastatic cells. Knockdown of KLF5 inhibited expression of many key genes in the pathway of actin cytoskeleton regulation, whereas overexpression of KLF5 elicited contrast effects (Fig. 3 G, H and Supplementary Fig. S3 G, H). Aligned with in silico analysis, depletion of KLF5 interrupted the dynamics of actin networks and induced unstructured actin polymerization, attenuated lamellipodia growth and unpolarized cell shapes (Fig. 3 I-K). Accordingly, control cells plated on 2D surfaces generated denser and more branched cortical actin filament arrays in cytoplasm and lamellipodia at the leading edge, forming larger F-actin-rich lamellipodia and increasing cell spreading area (Fig. 3 I-K). Taken together, these results demonstrate that KLF5-dependent actin branching drives lamellipodia growth and cell spreading, enhancing the cell motility in vitro. KLF5 preferentially occupies distal enhancer regions Given the canonical role of KLF5 in interpreting the genome, we employed calibrated chromatin immunoprecipitation sequencing (ChIP-seq) analysis to estimate the sequence preference and genomic binding loci of KLF5. To depict KLF5-related epigenetic landscape of NPC cells, we mined genome-wide profiles of NPC cell assessed by ChIP-seq (GSE95749) and analyzed distinct signal distribution pattern of epigenetic marks in the vicinity of KLF5 peaks. Based on signal of chromatin marks in C666-1 cells, the regions of KLF5 peaks were divided into three clusters: promoter (10.7%), enhancer (74.6%) and other regions (without marks, 14.7%) (Fig. 4 A). Clusters of KLF5 peaks displayed similar distribution in HK1 cells (Supplementary Fig. S4A). Most KLF5-occupied promoter and enhancer regions extensively overlapped with accessible regions inferred by assay for transposase-accessible chromatin (ATAC), suggestive of their accessibility to transcription factors and related collaborators (Fig. 4 A, B and Supplementary Fig. S4B). Among grouped KLF5 peaks, H3K4me1 indicated enhancer, H3K4me3 indicated promoter and H3K27ac indicated enhancer and promoter in all regions, respectively (Supplementary Fig. S4C-E). In H3K27ac + regions, the H3K4me3:H3K27ac ratio was higher than H3K4me1:H3K27ac in promoter cluster, whereas enhancer cluster exhibited contrast manifestation and other undefined regions exhibited no significant difference (Fig. 4 C). While location of KLF5 peaks annotated to specified genomic regions, we observed similar effect that H3K4me1:H3K27ac was higher in intergenic regions and gene body, and H3K4me3:H3K27ac was higher in promoters (Fig. 4 C). Approximately 15% of KLF5 peaks were located within compacted chromatin lacking ATAC signals (Fig. 4 A, B). The metagene analysis showed that H3K27ac, H3K4me1 and H3K4me3 modifications exhibited bimodal distribution pattern around KLF5 peak summit in enhancer or promoter cluster, while ATAC signal exhibited unimodal distribution (Fig. 4 B and Supplementary Fig. S4A). These results suggested that KLF5 occupancy may propel nucleosome removal or further DNA unwrapping, rendering relevant DNA surfaces accessible to KLF5 and other DNA-binding proteins. To examine occupancy preference of KLF5 at cis -regulatory elements and genomic regions, we grouped KLF5 peaks into quartiles based on the peak height and analyzed the distribution of KLF5 peaks relative to annotated genes. Overall, only 22% of KLF5 peaks were situated in proximity to (± 1kb) transcriptional start site (TSS), whereas 47% of KLF5 peaks were further than 10 kb to the closest TSS, and the proportion of peaks adjacent to TSS was inversely proportional to KLF5 affinity to genome (Fig. 4 D). Most (74.6%) KLF5 peaks occurred in enhancers that probably most were distal enhancers while KLF5 weakly bound most (73.3%) promoters (Fig. 4 E). Consistently, KLF5 preferred occupying distal regions (intergenic and gene body regions) instead of promoters, and the preference increased with the strength of KLF5 binding (Fig. 4 F, G and Supplementary Fig. S4F). To further characterize the KLF5 binding pattern in genome, we grouped KLF5 peaks into four fractions depending on H3K4me1:H3K27ac and H3K4me3:H3K27ac ratio. Among four quadrants, quadrant II (H3K4me1:H3K27ac+, H3K4me3:H3K27ac-) contained largest proportion of enhancers (32%) and distal regions (33%), while quadrant IV (H3K4me1:H3K27ac-, H3K4me3:H3K27ac+) contained most promoters (57%), supporting the notion that H3K4me1 marks enhancers and H3K4me3 marks promoters (Fig. 4 H and Supplementary Fig. S4G). Quadrant IV contained most (27%) Q1 KLF5 peaks, quadrant II contained most (29–30%) Q2 and Q3 KLF5 peaks, and quadrant I contained most (69%) Q4 KLF5 peaks, respectively (Fig. 4 I). Taken together, these findings uncover the previously undetermined pattern that KLF5 preferentially occupies the distal enhancer regions rather than promoters to regulate transcription. KLF5 transcriptionally activates ACTN4 by occupying its enhancer To identify the prominent target transcriptionally activated by KLF5 during cell migration, we analyzed the biological function of KLF5 cis -acting targets inferred by ChIP-seq through GO and KEGG pathway enrichment analysis, and found these genes implicated actin cytoskeleton regulation, lamellipodia formation and cell migration, which was consistent with analysis on KLF5-grouped transcriptome of NPC cells and samples (Fig. 5 A and Fig. 3 A-F). Among candidates involved in actin cytoskeleton regulation (ACTN4, ITGB5, VCL, VAV2, BAIAP2), alpha-actinin 4 (ACTN4), a protein engaging with dynamics of actin filaments [ 31 – 34 ], proficiently recruited KLF5 to its gene body (Fig. 5 B). With visualization of ChIP-seq data of KLF5 and histone modifications and ATAC-seq data in NPC cells on UCSC genome browser, we observed that, in ACTN4 gene body, enhancer marks (H3K4me1 and H3K27ac) and ATAC signal was enriched in the KLF5-binding region with relatively low H3K4me3 signal (Fig. 5 C and Supplementary Fig. S5A). Notably, KLF5 ChIP-seq for cells with various inherent characteristics uncovered similar binding peaks in ACTN4 gene body (Supplementary Fig. S5B), ranging from squamous cell carcinoma (SCC), gastric cancer (GC), colorectal cancer (CRC) to pancreatic ductal adenocarcinoma (PDAC). Simultaneously, the KLF5-binding region showed similar chromatin marks distribution and DNase I hypersensitivity in diverse cell types (Supplementary Fig. S5C), indicating this region as a conserved ACTN4 enhancer captured by KLF5 and recruitment of KLF5 to ACTN4 enhancer is a pervasive and heretofore undefined phenomenon. To determine the dominant KLF5 binding loci in the ACTN4 enhancer, we discovered top six consensus motifs enriched in KLF5 peaks (Fig. 5 D and Supplementary Fig. S5D). Since ACTN4 enhancer contained two potential motifs for KLF5 binding, we hypothesized that KLF5 binds ACTN4 enhancer at these two motifs and transcriptionally activates ACTN4 . To address it, we first generated KLF5 protein models by multiple tools, of which the best model predicted by Phyre2 was used for subsequent HDOCK molecular docking [ 35 , 36 ]. In KLF5-motif docking models (confidence score: 0.91, 0.86), ZnFs of KLF5 contacted the DNA major groove by hydrogen bond interactions between specific residues and bases (Supplementary Fig. S6A-C). In KLF5-motif1 model, residues Y384, S387, K391 and T396 of ZnF1 and W412 of ZnF2 contacted bases C2, G5’, G7’, C12, respectively (Supplementary Fig. S6B). In KLF5-motif2 model, residues T396 of ZnF1 and S445 and R454 of ZnF3 contacted bases G1’, G6’, C3, respectively (Supplementary Fig. S6C). The specific residues contacting ACTN4 enhancer overlapped with a transcriptional activation domain of KLF5 identified recently [ 37 ] (Supplementary Fig. S6A). Given the high docking confidence, we confirmed that endogenous KLF5 bound the two motifs in ACTN4 enhancer and another motif in ACTN4 promoter as evaluated by ChIP-qPCR (Fig. 5 E, F and Supplementary Fig. S6D). The two motifs in ACTN4 enhancer were marked by H3K27ac as well (Supplementary Fig. S6E-G). For orthogonal enhancer activity assays, we generated a luciferase reporter incorporating ACTN4 promoter and reporters incorporating two tandem gene cassettes: ACTN4 promoter followed by full-length or truncated ACTN4 enhancer with one motif. Compared to pGL3-Basic reporter, introduction of ACTN4 regulatory sequences substantially activated luciferase transcription, with strongest activation by full-length ACTN4 enhancer (Fig. 5 G, H and Supplementary Fig. S6H, I). Exogenous KLF5 further enhanced the luciferase activity (Fig. 5 G, H and Supplementary Fig. S6H, I). We next mutated both two motifs and found diminished luciferase activity upon the mutant (Fig. 5 I and Supplementary Fig. S6H, I), indicating that KLF5-bound loci dominate transcriptional activation ability of the ACTN4 enhancer. Analysis of Hi-C data of human umbilical vein endothelial cells (HUVEC) in a 3D-genome Interaction Viewer and database [ 38 ] showed the direct interplay between ACTN4 enhancer and ACTN4 promoter (Supplementary Fig. S7). These results together demonstrate that KLF5 occupies ACTN4 enhancer and facilitates ACTN4 enhancer-promoter interplay to activate ACTN4 transcription, potentially through chromatin looping or compartmentalization. ACTN4-actin conjunction enhances lamellipodia formation and cell motility Since KLF5 transcriptionally activated ACTN4 in NPC cells, the positive correlation between KLF5 and ACTN4 expression was confirmed in NPC samples (Fig. 6 A, B and Supplementary Fig. S8A, B). ACTN4 was upregulated in NPC samples, analogous to KLF5 expression pattern (Supplementary Fig. S8C). As opposed to ACTN4 low group, ACTN4 high group matched higher potential for metastatic progression (Fig. 6 C and Supplementary Fig. S8D). GSEA analysis revealed the metastasis relevant gene set enrichment in ACTN4 high group (Fig. 6 D), suggesting that ACTN4 engages metastatic pathways in NPC patients. We next knocked down ACTN4 in NPC cells, which mimicked KLF5 -silenced metastatic phenotypes of inferior capacity to migrate and invade in short-term migration and wound healing assays in vitro (Fig. 6 E-H and Supplementary Fig. S8E-G), indicating that ACTN4 imparts enhanced metastatic potential to NPC cells. Accordingly, overexpression of ACTN4 conveyed augmented metastatic activity (Fig. 6 I, J and Supplementary Fig. S8H-J). Introduction of ACTN4 partially rescued the impaired metastatic activity of KLF5 -KD NPC cells (Fig. 6 K, L and Supplementary Fig. S8K, L). In view of our data showing that KLF5 regulated actin remodeling and lamellipodia formation, as well as the well-established role of ACTN4 in actin dynamics and cell motility [ 31 – 34 ], we speculated that ACTN4, transcriptionally activated by KLF5, crosslinks with actin filaments, triggering denser lamellipodial actin networks and lamellipodia growth. We next applied GSEA analysis of the expression profiles from ACTN4 high and ACTN4 low NPC groups, which revealed an active state of actin networks and lamellipodia organization at the cell leading edge underlying faster cell migration in ACTN4 high NPC group (Supplementary Fig. S8M). We further performed immunofluorescence analysis to detect the distribution of ACTN4 and actin filaments in ACTN4- overexpressed NPC cells. ACTN4 colocalized with F-actin but not with dissociative actin in the whole cell (Fig. 7 A). Intriguingly, exogenous ACTN4 induced accumulation of actin filaments in lamellipodia rather than in the whole cell (Fig. 7 A, B). Moreover, larger area of lamellipodia were detected in cells with exogenous ACTN4, enhancing cell spread on 2D surface (Fig. 7 A, B). Radial line profile analysis showed that, F-actin intensively localized throughout the extending lamellipodia at the cell edge, and ACTN4 displayed similar distribution pattern that the fluorescence intensity increased with distance from nucleus (Fig. 7 C). In line with previous study [ 31 ], exogenous ACTN4 substantially colocalized with highly branched and dense actin filament networks, especially at the cell leading edge and membrane lamellipodia (Fig. 7 C). Colocalization analysis corroborated the extensive colocalization between ACTN4 and F-actin in HONE-1 (Pearson’s correlation value: 0.8; Manders’ colocalization coefficients: tM1 = 0.908 and tM2 = 0.901) and SUNE-1 cells (Pearson’s correlation value: 0.7; Manders’ colocalization coefficients: tM1 = 0.836 and tM2 = 0.819). Knockdown of ACTN4 impaired the ACTN4-actin conjunction and formation of branched actin networks, causing defective cell morphology and lamellipodia extending (Fig. 7 D). Collectively, these results demonstrate the critical role of ACTN4 in remodeling actin networks at the cell edge to facilitate lamellipodia formation. Discussion In this study, we elucidate that KLF5 is a master TF that preferentially occupies the ACTN4 enhancer to activate its transcription, which regulates the formation of actin-driven lamellipodia and enhances the intrinsic metastatic potential of NPC cells in vitro and in vivo (Fig. 8 ). Through analyzing transcriptional interactome and regulatory network, we identified KLF5 as a master TF governing transcription program in NPC metastasis and exacerbation. Emerging evidence reveals that KLF5 promotes cancer metastasis under diverse contexts through divergent pathways [ 39 – 41 ]. Our analysis suggests that NPC patients bearing high KLF5 burden are prone to develop distant metastasis and progress to an advanced stage. By systematically analyzing the KLF5-related epigenetic landscape assessed by ChIP-seq and ATAC-seq, we discover the previously undetermined phenomenon that a large group of KLF5 peaks (74.6%) occur at distal regions coinciding with enhancer cis -regulatory elements in NPC, indicating its occupancy preference for distal enhancer regions. Enrichment of H3K27ac and ATAC signal in KLF5-captured promoters and enhancer regions reveals the chromatin accessibility and transcription active state of these regions associated genes. Accumulating evidence has linked KLF4 with dynamic enhancer organization which directs spatiotemporal gene expression programs in pluripotent stem cells [ 21 , 42 , 43 ]. Previous chromatin profiling studies showed that KLF4 cooperating with reprogramming factors mainly occupies enhancers in reprogramming and established pluripotent stem cells [ 43 ]. KLF4 orchestrates the long-range chromatin loops rendering physical contacts between enhancers and promoters through interaction with architectural proteins such as cohesion [ 21 , 42 ]. Recruitment of pioneer factors to chromatinized binding sites opens and remodels the chromatin and facilitates subsequent binding of other TFs and cofactors [ 44 , 45 ]. KLF4 is a pioneer factor promoting reprogramming and the impaired pluripotency due to loss of KLF4 can be compensated by other KLFs (e.g., KLF5), indicating potential pioneer factor identity of them [ 46 , 47 ]. Given the bimodal distribution of histone marks and unimodal distribution of ATAC signal around KLF5 peak summit, whether KLF5 operates as a pioneer factor and reads out nucleosomal DNA in compacted chromatin to facilitate appropriate nucleosome occupancy and position needs further study. Orchestrated by transcription modulation, cell migration predominantly depends on precise tuning of global actin flow at the cell front and rear [ 6 , 48 ]. Our analysis shows that KLF5 interprets the genome to respond to promigratory cues, triggering rapid modulation of genes involved in actin cytoskeleton transcriptionally. Among these candidates, ACTN4 proficiently recruits KLF5 to its gene body. Integrating ChIP-seq data of chromatin marks and KLF5 under various contexts, we discovered a heretofore unrecognized and conserved enhancer element in ACTN4 gene body occupied by KLF5. Intriguingly, introducing truncated ACTN4 enhancer with one motif slightly impaired the transcriptional activation strength of ACTN4 promoter with or without ectopic KLF5, presumably attributed to sequence integrity requirement for stabilizing the enhancer-promoter interplay. Alternatively, the truncated ACTN4 enhancer lacks potential to recruit sufficient KLF5 and co-binding transcription activators capturing flanking sequences, hence some repressive TFs and corepressors partially substitute for KLF5. Given canonical regulatory modes of long-range enhancer-promoter crosstalk [ 49 – 51 ], KLF5 on ACTN4 enhancer may recruit coactivators and promote the chromatin loop extrusion or genome compartmentalization to synergistically drive transcription. While the structure of KLF5-DNA complex remains undetermined, we simulated the docking of putative KLF5 structure and its binding motif in ACTN4 enhancer inferred by ChIP-seq and found a high confidence of the KLF5-motif docking model. Additional essential biochemical information will be obtained by high-resolution structure research of purified KLF5 and its partners. Recent comprehensive measurements of human transcriptional effector domains revealed that KLF5 boasts activation and repression domains, rendering the bifunctional potential of KLF5 in specific contexts [ 37 ]. Thus, in response to distinct cues, KLF5 potentially oscillates between activating and repressing activity at various genomic loci and the regulatory output can be delineated by stoichiometries. The transcription activation of ACTN4 by KLF5 explains the impaired actin network structure, actin-rich lamellipodia, cell spreading and directed migration caused by depletion of KLF5 in NPC cells. ACTN4-actin conjunction empowers metastatic NPC cells to generate actin-driven lamellipodia and efficiently migrate faster to seek congenial milieu to survive and grow. Though studying KLF5-ACTN4 facilitated cell migration using 2D systems reveals the mechanism of how KLF5 enhances the metastatic potential of cells in vitro and in vivo, utilization of tools and more complex systems in more physiological contexts can deepen our understanding of migration principles underlying metastasis, through which providing promising therapeutic avenues to control metastasis — the overwhelming cause of cancer-associated death. To prevent metastasis and eliminate established metastatic lesions, numerous agents have been developed to identify and drug targets in metastatic cascade, of which TFs are switching from ‘undruggable’ to ‘druggable’ [ 52 – 54 ]. However, clinical validation of TF-targeting agents and incorporating them into the standard of care remains problematic, owing to their cytostatic manifestation in preclinical models and compensatory pathways involving requisite steps in metastasis [ 52 ]. Therefore, rational combination therapies are efficacious in targeting metastasis. KLF5, the dysregulated master TF in metastatic NPC, represents a unique potential therapeutic target controlling metastasis, with challenges of developing inhibitors targeting its protein-DNA and protein-protein interplay [ 54 ]. Though inhibitors targeting KLF5-DNA binding remain to be developed, a recent effort overcame the difficulty of inhibitor recognizing convex and highly positively charged DNA binding interfaces [ 54 , 55 ]. An agent targeting RUNX-DNA binding increased survival of mouse xenograft models [ 55 ]. Inhibitors interrupting binding of KLF5 on ACTN4 enhancer and other loci may serve as means to mitigate metastases of NPC. Moreover, utilizing the induced proximity principle and ubiquitin-proteasome system, the proteolysis-targeting chimaera molecules potentially degrade KLF5 through a ligand binding KLF5 and another ligand recruiting ubiquitin ligases [ 56 ]. In summary, our discoveries underscore the prometastatic role of KLF5, a master TF in NPC, and provide mechanistic insights to identify potential biomarkers and therapeutic interventions to shrink metastases. Novel agents targeting mutated or dysregulated KLF5 and other master TFs in combination with first-line therapies will improve outcome of patients with metastatic disease. Materials and Methods Transcriptional interactome and regulatory network inference The transcriptional interactomes were generated by ARACNe-AP algorithm [ 26 ] from 4 GEO datasets profiled by RNA-seq or microarray: GSE118719, GSE68799, GSE13597 and GSE103611. ARACNe-AP was run with 100 reproducible bootstrap iterations using retrieved gene expression profiles and 1,639 TFs as predefined input, with parameters setting to no DPI (Data Processing Inequality) tolerance and MI (Mutual Information) p -value threshold of 10 − 8 . Inferred significant KLF-target interactions were filtered ( P < 0.05) for subsequent analysis. With input of transcriptional interactomes and gene expression profiles, regulatory networks were reverse engineered to output regulons. TFs activity inference TFs activity profiles were estimated by msVIPER algorithm [ 28 ], which tests regulon enrichment on gene expression signatures. By comparing NPC samples under specific contexts (e.g., metastatic versus non-metastatic), gene expression signatures were generated to identify potential role of TFs in NPC progression. After randomly permuted samples of gene expression profiles 1,000 times, null models were produced by using signatures generated with permutation iterations. Comparing each regulon enrichment score to a null model, TFs activity was inferred as normalized enrichment score, facilitating the identification of master TFs in distant metastasis of NPC. Cell cultrure Six human NPC cell lines (C666-1, CNE1, CNE2, HNE-1, HONE-1 and SUNE-1) were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; ExCell Bio). Human immortalized normal nasopharyngeal epithelial cell line (N2-Tert) was maintained in keratinocyte serum–free medium (Invitrogen), supplemented with bovine pituitary extract (BD Biosciences). The above cell lines were authenticated and generously provided by M.S. Zeng (SYSUCC, China). Human Embryonic Kidney 293T (HEK293T) cells obtained from the ATCC was maintained in DMEM (Gibco) supplemented with 10% FBS (ExCell Bio). RNA extraction, RT-PCR and qPCR Total RNA was extracted from cells using either TRIzol reagent (Invitrogen) or RNA Quick Purification kit (ESscience) according to the manufacturer’s instructions, following RNA quality and quantity measurement by NanoDrop 2000 (Thermo Fisher Scientific). cDNA was generated using HiScript III RT SuperMix for qPCR (Vazyme) according to the manufacturer’s instructions. cDNA was diluted 1:10 in distilled water and 2 µl was used per qPCR reaction. qRT-PCR was performed using SYBR qPCR Master Mix (Vazyme) on CFX96 Touch sequence detection system (Bio-Rad). Primers were designed using the Primer-BLAST tool at NCBI. Housekeeping gene mRNA level (GAPDH) was used for normalization. The mRNA levels of all genes were quantified using the 2 −ΔΔCt method to infer the difference. qPCR primer sets are listed in Supplementary Table S3 . Western blotting Cells were lysed on ice using the radioimmunoprecipitation assay (RIPA, Merck Millipore) supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland) for 30 min and centrifuge at 4°C for 15 min at 12,000 rpm. The protein concentrations of supernatants were determined using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Total protein lysates (30 µg) were separated by SDS–polyacrylamide gel electrophoresis (4–20%) and transferred to PVDF membranes (Merck Millipore). Membranes were blocked for 1 h in 5% skimmed milk and then incubated overnight at 4°C in primary antibody solution. After washing in TBST, membranes were incubated with secondary antibodies for 1h at room temperature. Proteins were detected using Super ECL Plus (Applygen). The following primary antibodies were used: anti-KLF5 (rabbit; Sigma-Aldrich, 09822, 1:2000), anti-HA (rabbit; Abcam, ab9110, 1:10000), anti-ACTN4 (rabbit; Abcam, ab108198, 1:5000), anti-GAPDH (rabbit; Abcam, ab181602, 1:10000), anti-alpha Tubulin (mouse; Abcam, ab7291, 1:10000). Transient transfection and stable cell line construction For siRNA-mediated RNA interference, siRNAs targeting human KLF5 and non-targeting control were synthesized and ordered from GenePharma (Suzhou, China). Human KLF5 or ACTN4 CDS region cDNA tagged by HA was cloned into the pSin-EF2-Puro plasmid (Addgene) using BamHI and EcoRI restriction sites. The siRNAs or plasmids were transfected to cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. The shRNAs targeting human KLF5 and ACTN4 or non-targeting control were cloned into the pLKO.1-Puro plasmid (Addgene) using AgeI and EcoRI restriction sites. Lentiviral particles were generated by transfecting pLKO.1-shRNA or pSin-EF2-cDNA and psPAX2 plasmid (Addgene) and pMD2G plasmid (Addgene) in HEK293T cells with polyethyleneimine (PEI; Polysciences). Lentiviral supernatants were harveseted 48 h after transfection and were filtered by a 0.45 µm pore size PVDF membrane (jet). HONE-1 and SUNE-1 cells were infected by lentiviruses with polybrene (Beyotime). After 24 h infection, stable cell lines were selected by puromycin for 72 h and verified by western blotting. A list of targeted sequences for siRNA and shRNA is provided in Supplementary Table S4 and 5. Cell viability and colony formation assays For the cell viability assay, 1,000 cells were seeded to each well of 96-well plates (jet) in 10% FBS-added medium and cultured for 5 days. After incubated for 12 h, cell viability was measured by a CCK-8 kit (APExBIO) according to the manufacturer’s instructions and measured every 24 hours after first measurement. Absorbance at 450 nm was recorded by a spectrophotometric plate reader (BioTek ELX800, Bio-Rad). For colony formation assay, 400 cells were seeded to each well of six-well plates (jet) in 10% FBS-added medium and cultured for 10–14 days. Detectable colonies were fixed with methanol for 10 min, stained with hematoxylin for 1 h and analyzed. Wound healing assay Cells were seeded into six-well plates (jet) and cultured to a 100% confluency, following a scratch using a 200 µl pipette tip and a plastic ruler as a guide in the cell monolayers. Detached cells were washed by phosphate-buffered saline (PBS), after which images were captured by the inverted microscope (NIKON Eclipse Ti2-U). Remaining cells were allowed for 24 h growing and were imaged with the inverted microscope (NIKON Eclipse Ti2-U). Images were analysed using Fiji/ImageJ by drawing a line indicating the migrating fronts on two sides of the scratch wound and comparing healing rate between groups. Transwell migration and invasion assays For Transwell migration and invasion assays, 8 µm pore size transwell chambers (corning) were coated with or without Matrigel (BD Biosciences) according to the manufacturer’s instructions. Cells resuspended in 200 µl serum-free medium were seeded into the upper chambers with 3.5 × 10 4 HONE-1 cells or 4.5 × 10 4 SUNE-1 cells for migration assay and 7 × 10 4 HONE-1 cells or 9 × 10 4 SUNE-1 cells for invasion assay. Lower chambers were filled with 500 µl 10% FBS-added medium. After incubation for 12–14 hours (migration assay) or 20–22 hours (invasion assay), the migrated or invaded cells were fixed with methanol for 10 min, stained with hematoxylin for 2 h and imaged by inverted microscope (NIKON Eclipse Ti2-U). Cell migration and invasion capability were defined as relative number of migrated or invaded cells manually counted. In vivo metastasis models In vivo metastasis experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Ethics Committee of SYSUCC (L025504202207003). BALB/c nude mice (female, 4–6 weeks old, 15g) were obtained from Charles River Laboratories (Beijing, China) and maintained at the Animal Experiment Center of the Sun Yat-sen University. For the lung metastasis model, KLF5 -KD or control HONE-1 cells (1 × 10 6 cells) in PBS were injected into the tail vein of mice (n = 6 per group). One month after injection, mice were euthanized and their lungs were dissected and analyzed for metastasis. For the inguinal lymph node metastasis model, KLF5 -KD or control HONE-1 and SUNE-1 cells (2 × 10 5 cells) in PBS were inoculated into the footpads of mice (n = 6 per group and n = 10 per group). One month after inoculation, mice were euthanized and their footpad primary tumors and inguinal tumor-draining lymph nodes were dissected and analyzed for metastasis. The primary tumors and lungs were fixed in 4% formaldehyde for 48 h. Tissues were embedded in paraffin, sectioned into 5 µm pieces, and mounted on slides for hematoxylin and eosin staining. Slides were imaged using an Olympus scanning system (VS200). Immunofluorescence and image analysis For Immunofluorescence assay, cells were fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100 and blocked in 3% BSA for 1h at room temperature. Cells were incubated at 4°C with an anti-HA antibody (rabbit; ab9110; 1:1000) diluted in 1% BSA solution overnight. After washing three times with PBS, cells were incubated for 1 h at room temperature with Alexa Fluor 594 goat anti-rabbit IgG H&L (abcam; ab150080; 1:500). diluted in 1% BSA solution. Nuclei and F-actin were then stained with DAPI (Biosharp) and phalloidin-Alexa Fluor 488 (Beyotime), respectively. After washing three times with PBS, cells were mounted using antifade mounting medium (Beyotime). Cells were imaged using a 63× oil-immersion objective on a Zeiss confocal system (LSM 980). Mean intensity of F-actin and spreading area in the whole cell and lamellipodia were recorded through manually drawing the free-form regions of interest (ROI) using Fiji/ImageJ. For radial line profile analysis, line scans of captured images were generated through lamellipodial outlines and the cell edge was applied as a reference to evaluate the spatial distribution of ACTN4 and F-actin. The Pearson correlation coefficient (r) was calculated between the mean intensity of F-actin and ACTN4 at different distance. For colocalization analysis, images of cells expressing ectopic ACTN4 were exported to Fiji and colocalization efficiency and specificity of ACTN4 and F-actin was analyzed using the Fiji Coloc2 plugin. ChIP ChIP assay was performed using Pierce Magnetic ChIP Kit (26157, Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, 4 × 10 6 cultured HONE-1 or SUNE-1 cells were harvested and washed with ice-cold PBS twice. Cells were fixed in 2ml of 1% formaldehyde for 10 min at room temperature, and Glycine Solution (10×) was added to a final concentration of 1× to quench crosslink for 5 min at room temperature, following two washes with ice-cold PBS. Cells were collected by mechanically scrapping in 1mL of ice-cold PBS with 10 µL of the Halt Cocktail and centrifuged at 3,000 × g for 5 min at 4°C. After removing PBS, crosslinked cells were lysed using Membrane Extraction Buffer containing protease/phosphatase inhibitors and digested using MNase. After sonication on ice to break nuclear membrane, solutions were centrifuged at 9,000 × g for 5 min at 4°C. 10% of the supernatants containing the digested chromatin was stored as input sample, and the remaining supernatants were incubated with primary antibodies: 1 µL IgG and 3–5 µg anti-KLF5 (rabbit; Sigma-Aldrich, 09822), anti-HA (rabbit; Abcam, ab9110) or anti-H3K27ac (rabbit; ab177178) at 4°C overnight with mixing. Protein A/G Magnetic Beads were added and incubated for 2 hours at 4°C with mixing. Beads were collected with a magnetic stand and were washed three times with IP Wash Buffer 1 and once with IP Wash Buffer 2. The protein-DNA complexes were eluted from beads and de-crosslinked using IP Elution Buffer containing NaCl and Proteinase K for 3 hours at 65°C. DNA samples were then purified and subjected to qPCR or ChIP-seq library generation. Primers were provided in Supplementary Table S6. Dual-luciferase reporter assay Promoters of ACTN4 (1,000 bp upstream of the transcription start site) and truncated ACTN4 enhancer containing motif1/2 or full-length ACTN4 enhancer or mutant ACTN4 enhancer (all bases of motifs replaced with adenosines) were cloned into pGL3-basic luciferase reporter plasmid (Promega). Inserted sequences were provided in Supplementary Table S7. Cells were seeded into 24-well plates and co-transfected with plasmids encoding an empty vector or HA-tagged KLF5 (400 ng) and luciferase reporter plasmids (100 ng) and a Renilla luciferase reporter (Addgene, 10 ng) using Lipofectamine 3000 (Invitrogen). Cells were lysed after 24-48h of transfection by Dual-Luciferase Reporter Assay System (E1910, Promega) following the manufacturer’s instructions. Luciferase activity was measured by GloMax Navigator (Promega), and firefly luciferase activity was normalized to Renilla luciferase activity. Luciferase mRNA level in different groups were evaluated through RNA extraction, RT-PCR and qPCR. ChIP-seq The ChIP-seq libraries were prepared using Paired-End DNA Sample Prep kit (Illumina) according to the manufacturer’s instructions. The libraries were amplified by PCR with 18 cycles and size selected for 100-300bp fragments. Qualified libraries were sequenced on a NextSeq 500 Sequencer (Illumina) according to the manufacturer’s instructions. Raw reads were filtered and quality-verified using SOAPnuke program (v.2.0). After filtering, the clean data was mapped to human genome GRCh38 by SOAP2 (v.2.21). Peaks were called using model-based MACS2 software (v.2.1.1) and annotated with the closest hg38 genes by annotatePeaks of HOMER (v.4.11). Comparing peaks from two ChIP-seq libraries (IgG and KLF5 groups), KLF5-unique peaks were identified by MAnorm (v.1.2.0), which met the standard that |M| >= 1 and P value < = 10 − 5 . The ChIP-seq data for this study are available for download from the Gene Expression Omnibus (GEO) repository (GSE243951). Analysis and visualization of ChIP-seq, ATAC-seq and Hi-C data The following ChIP-seq and ATAC-seq data were downloaded from the publicly available datasets: GSE95749 (NPC), including C666-1_H3K4me1, C666-1_H3K4me3, C666-1_H3K27ac, C666-1_ATAC, HK1_H3K4me1, HK1_H3K4me3 and HK1_H3K27ac; GSE88976 (SCC), BICR31_KLF5; GSE51705 (GC), KATOIII_KLF5; GSE49402 (CRC), LoVo_KLF5; GSE64557 (PDAC), CFPAC-1_KLF5. The genome reference of public ChIP-seq and ATAC-seq data were converted from human hg19 to hg38 using CrossMap (v.0.5.4). ChIP-seq and ATAC-seq identified peaks and normalized signal on gene tracks were visualized using UCSC Genome Browser. KLF5 and chromatin marks occupancy on genomic regions were created using the computeMatrix and the plotHeatmap tools of deepTools (v.3.5.2). Based on signal of chromatin marks, KLF5 ChIP-seq regions were categorized into three clusters: promoter regions (H3K27ac and H3K3me3-enriched), enhancer regions (H3K27ac and H3K3me1-enriched) and other undefined regions. Metaplots revealing KLF5 and chromatin marks enrichment in different regions were created using the plotProfile tool of deepTools (v.3.5.2). ChIP-seq and ATAC-seq identified peaks were annotated to specified genomic regions (promoter, peaks occurring at ± 1kb intervals around the hg38 gene transcription start site; gene body, peaks occurring at gene bodies; intergenic, peaks occurring outside promoters and gene bodies) using ChIPseeker (v.1.28.3). Analysis of a Hi-C contact map in HUVEC cell was performed using a 3D-genome Interaction Viewer and database (3DIV) to generate chromatin interactions between ACTN4 promoter and ACTN4 enhancer. Motif enrichment analysis For motif discovery, the motifs in KLF5 peaks of ChIP-seq were discovered using MEME (v.4.10.1) with the significance cutoff E value < 10 − 8 . For motif comparison, the discovered motifs in KLF5 peaks of ChIP-seq were compared to known motifs in HOCOMOCOv11 database using Tomtom (v.5.5.3). RNA-seq analysis For RNA-seq analysis of HONE-1 cells with or without knockdown of KLF5 , total RNA was extracted from cells using TRIzol reagent (Invitrogen). Purified mRNA using Dynabeads Oligo (dT) (Thermo Fisher) was fragmented by NEBNext Magnesium RNA fragmentation module (NEB) and reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen), following U-labeled second-stranded DNAs synthesis with E. coli DNA polymerase I (NEB), RNase H (NEB) and dUTP Solution (Thermo Fisher). The final cDNA libraries were constructed by random hexamer primed cDNA synthesis, PCR amplification and size selection as 300 ± 50 bp. The cDNA libraries were sequenced on the Illumina Novaseq™ 6000 platform according to the manufacturer’s instructions. Raw reads were filtered and quality-verified using cutadapt (v.1.9.1) and FastQC (v.0.11.9) before alignment. Reads were aligned and assembled using HISAT2 (v.2.2.1) and StringTie (v.2.1.6) against the human genome GRCh38. Transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare software (v.0.9.8). Transcript-level and gene-level were quantified using StringTie and ballgown. Normalization of raw count data and genes differential expression analysis were performed using the DESeq2 software. The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 were considered differentially expressed genes (DEGs), subjected to following GO, KEGG and GSEA analysis. The RNA-seq data for this study are available for download from the Gene Expression Omnibus (GEO) repository (GSE243952). GO and KEGG analysis GO and KEGG enrichment analysis were performed against Human MSigDB (v2023.1. Hs) collections c2 (KEGG subset of Canonical pathways) and c5 (Gene Ontology gene sets) for genes ranging from DEGs from RNA-seq of HONE-1 cells with or without knockdown of KLF5 , DEGs identified from KLF5 hi and KLF5 lo groups (categorized by KLF5 median expression level) in GSE13597 and GSE103611 datasets to KLF5 cis -acting candidates in ChIP-seq. GO terms and pathways meeting this condition with p < 0.05 were defined as significantly enriched GO terms and pathways in DEGs. Gene set enrichment analysis (GSEA) Gene set enrichment analysis was performed using GSEA (v4.1.0) with default parameters against Human MSigDB (v2023.1. Hs) collections c2 (KEGG subset and Reactome subset of Canonical pathways), c5 (Gene Ontology gene sets), metastasis-associated gene sets and hallmark gene sets. Differentially enriched gene sets were analyzed for KLF5 -KD and control RNA-seq dataset and GEO datasets which were grouped into KLF5 hi and KLF5 lo groups or ACTN4 hi and ACTN4 lo groups. |NES|>1, NOM p -val < 0.05, FDR < 0.25 were considered to be different in two groups. Model generation of KLF5 and molecular docking simulation of KLF5- ACTN4 enhancer motif complex KLF5 protein models were generated using five specific systems based on homology modeling method: SWISS-MODEL, Phyre2, I-Tasser, Robetta and AlphaFold2 and one system based on Ab initio prediction method: trRosetta. The quality of models for each system were assessed by their own scoring functions and other assessment programs including ProQ, Verify3D, PROCHECK and SolVX for three criteria: spatial geometry quality, stereochemistry quality and energetical stability. The best model predicted by Phyre2 was used for subsequent HDOCK molecular docking with the two motifs in ACTN4 enhancer, respectively. The docking models with best docking score and confidence score were selected to perform molecular graphical analysis using UCSF ChimeraX (v.1.6.1). Statistical analysis Statistical analysis was performed by GraphPad Prism 9 (GraphPad Software). P value < 0.05 was considered to be statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For data with Gaussian distributions, statistical comparison was made using Two-tailed Student’s t -test between two groups and one-way ANOVA for more than two groups. Data represent the mean ± SD unless stated otherwise. Declarations Data availability All data generated or analyzed during this study are included in this article and its Supplementary Information Files. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (92259202, 82273401, 81872463), the Guangdong Basic and Applied Basic Research Foundation (2019A1515011863, 2019A1515012045, 2023B1515020014), the Health & Medical Collaborative Innovation Project of Guangzhou City, China (201803040003) and the Special Support Program of Sun Yat-sen University Cancer Center (16zxtzlc06). Author contributions GZ and DW designed and supervised the study. GZ, DW and ZY conducted the literature search. ZY, YP, YW and PY performed the experiments. ZY and XX performed the bioinformatic analysis. ZH developed the animal models. TQ, ZY and YP extracted and analyzed data. GZ, ZY, DW and YS were responsible for interpreting results. ZY, YW, DW and GZ contributed to the figures and tables. ZY and DW wrote the manuscript. Competing Interests The authors declare no competing interests. References Gerstberger S, Jiang Q, Ganesh K. Metastasis. Cell 2023; 186: 1564–79. Massagué J, Ganesh K. Metastasis-Initiating Cells and Ecosystems. Cancer Discov 2021; 11: 971–94. Follain G, Herrmann D, Harlepp S, Hyenne V, Osmani N, Warren SC et al. Fluids and their mechanics in tumour transit: shaping metastasis. Nat Rev Cancer 2020; 20: 107–24. SenGupta S, Parent CA, Bear JE. The principles of directed cell migration. Nat Rev Mol Cell Biol 2021; 22: 529–47. Fu A, Yao B, Dong T, Chen Y, Yao J, Liu Y et al. Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer. Cell 2022; 185: 1356–72. e26. Bera K, Kiepas A, Godet I, Li Y, Mehta P, Ifemembi B et al. Extracellular fluid viscosity enhances cell migration and cancer dissemination. Nature 2022; 611: 365–73. Hakala M, Wioland H, Tolonen M, Kotila T, Jegou A, Romet-Lemonne G et al. Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks. Nat Cell Biol 2021; 23: 147–59. Banerjee T, Biswas D, Pal DS, Miao Y, Iglesias PA, Devreotes PN. Spatiotemporal dynamics of membrane surface charge regulates cell polarity and migration. Nat Cell Biol 2022; 24: 1499–1515. Reversat A, Gaertner F, Merrin J, Stopp J, Tasciyan S, Aguilera J et al. Cellular locomotion using environmental topography. Nature 2020; 582: 582–5. Mehidi A, Kage F, Karatas Z, Cercy M, Schaks M, Polesskaya A et al. Forces generated by lamellipodial actin filament elongation regulate the WAVE complex during cell migration. Nat Cell Biol 2021; 23: 1148–62. De Belly H, Yan S, Borja da Rocha H, Ichbiah S, Town JP, Zager PJ et al. Cell protrusions and contractions generate long-range membrane tension propagation. Cell 2023; 186: 3049–61. e15. Bisaria A, Hayer A, Garbett D, Cohen D, Meyer T. Membrane-proximal F-actin restricts local membrane protrusions and directs cell migration. Science 2020; 368: 1205–10. Lappalainen P, Kotila T, Jégou A, Romet-Lemonne G. Biochemical and mechanical regulation of actin dynamics. Nat Rev Mol Cell Biol 2022; 23: 836–52. Liu X, Nie L, Zhang Y, Yan Y, Wang C, Colic M et al. Actin cytoskeleton vulnerability to disulfide stress mediates disulfidptosis. Nat Cell Biol 2023; 25: 404–14. Zou H, Poore B, Brown EE, Qian J, Xie B, Asimakidou E et al. A neurodevelopmental epigenetic programme mediated by SMARCD3-DAB1-Reelin signalling is hijacked to promote medulloblastoma metastasis. Nat Cell Biol 2023; 25: 493–507. Tetreault M-P, Yang Y, Katz JP. Krüppel-like factors in cancer. Nat Rev Cancer 2013; 13: 701–13. Chen Y, Lüttmann FF, Schoger E, Schöler HR, Zelarayán LC, Kim K-P et al. Reversible reprogramming of cardiomyocytes to a fetal state drives heart regeneration in mice. Science 2021; 373: 1537–40. Blacher E, Tsai C, Litichevskiy L, Shipony Z, Iweka CA, Schneider KM et al. Aging disrupts circadian gene regulation and function in macrophages. Nat Immunol 2022; 23: 229–6. Wang Z, Yang L, Wu P, Li X, Tang Y, Ou X et al. The circROBO1/KLF5/FUS feedback loop regulates the liver metastasis of breast cancer by inhibiting the selective autophagy of afadin. Mol Cancer 2022; 21: 9. Zeng L, Zhu Y, Moreno CS, Wan Y. New insights into KLFs and SOXs in cancer pathogenesis, stemness, and therapy. Semin Cancer Biol 2023; 90: 29–44. Di Giammartino DC, Kloetgen A, Polyzos A, Liu Y, Kim D, Murphy D et al. KLF4 is involved in the organization and regulation of pluripotency-associated three-dimensional enhancer networks. Nat Cell Biol 2019; 21: 1179–90. Sung 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. CA Cancer J Clin 2021; 71: 209–49. Ferlay J, Ervik M, Lam F, Colombet M, Mery L, Piñeros M, Znaor A, Soerjomataram I, Bray F. Global Cancer Observatory: Cancer Today. Lyon, France: International Agency for Research on Cancer. 2020. https://gco.iarc.fr/today . Accessed 2 Aug 2023. Zhang Y, Chen L, Hu G-Q, Zhang N, Zhu X-D, Yang K-Y et al. Gemcitabine and Cisplatin Induction Chemotherapy in Nasopharyngeal Carcinoma. N Engl J Med 2019; 381: 1124–35. Yang Y, Pan J, Wang H, Zhao Y, Qu S, Chen N et al. Tislelizumab plus chemotherapy as first-line treatment for recurrent or metastatic nasopharyngeal cancer: A multicenter phase 3 trial (RATIONALE-309). Cancer Cell 2023; 41: 1061–72. e4. Lachmann A, Giorgi FM, Lopez G, Califano A. ARACNe-AP: gene network reverse engineering through adaptive partitioning inference of mutual information. Bioinformatics 2016; 32: 2233–5. Lambert SA, Jolma A, Campitelli LF, Das PK, Yin Y, Albu M et al. The Human Transcription Factors. Cell 2018; 172: 650–65. Alvarez MJ, Shen Y, Giorgi FM, Lachmann A, Ding BB, Ye BH et al. Functional characterization of somatic mutations in cancer using network-based inference of protein activity. Nat Genet 2016; 48: 838–47. He P, Yang JW, Yang VW, Bialkowska AB. Krüppel-like Factor 5, Increased in Pancreatic Ductal Adenocarcinoma, Promotes Proliferation, Acinar-to-Ductal Metaplasia, Pancreatic Intraepithelial Neoplasia, and Tumor Growth in Mice. Gastroenterology 2018; 154: 1494–1508.e13. Mueller J, Szep G, Nemethova M, de Vries I, Lieber AD, Winkler C et al. Load Adaptation of Lamellipodial Actin Networks. Cell 2017; 171: 188–200.e16. Kaplan JM, Kim SH, North KN, Rennke H, Correia LA, Tong HQ et al. Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis. Nat Genet 2000; 24: 251–6. Wang M-C, Chang Y-H, Wu C-C, Tyan Y-C, Chang H-C, Goan Y-G et al. Alpha-actinin 4 is associated with cancer cell motility and is a potential biomarker in non-small cell lung cancer. J Thorac Oncol 2015; 10: 286–301. Huang Z, Zhou J-K, Wang K, Chen H, Qin S, Liu J et al. PDLIM1 Inhibits Tumor Metastasis Through Activating Hippo Signaling in Hepatocellular Carcinoma. Hepatology 2020; 71: 1643–59. Feng D, Kumar M, Muntel J, Gurley SB, Birrane G, Stillman IE et al. Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis. J Am Soc Nephrol 2020; 31: 1479–95. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 2015; 10: 845–58. Yan Y, Tao H, He J, Huang S-Y. The HDOCK server for integrated protein-protein docking. Nat Protoc 2020; 15: 1829–52. DelRosso N, Tycko J, Suzuki P, Andrews C, Aradhana, Mukund A et al. Large-scale mapping and mutagenesis of human transcriptional effector domains. Nature 2023; 616: 365–72. Kim K, Jang I, Kim M, Choi J, Kim M-S, Lee B et al. 3DIV update for 2021: a comprehensive resource of 3D genome and 3D cancer genome. Nucleic Acids Res 2021; 49: D38–D46. Zhang B, Li Y, Wu Q, Xie L, Barwick B, Fu C et al. Acetylation of KLF5 maintains EMT and tumorigenicity to cause chemoresistant bone metastasis in prostate cancer. Nat Commun 2021; 12: 1714. Liu P, Wang Z, Ou X, Wu P, Zhang Y, Wu S et al. The FUS/circEZH2/KLF5/ feedback loop contributes to CXCR4-induced liver metastasis of breast cancer by enhancing epithelial-mesenchymal transition. Mol Cancer 2022; 21: 198. Huang Q, Liu M, Zhang D, Lin B-B, Fu X, Zhang Z et al. Nitazoxanide inhibits acetylated KLF5-induced bone metastasis by modulating KLF5 function in prostate cancer. BMC Med 2023; 21: 68. Wei Z, Gao F, Kim S, Yang H, Lyu J, An W et al. Klf4 organizes long-range chromosomal interactions with the oct4 locus in reprogramming and pluripotency. Cell Stem Cell 2013; 13: 36–47. Chronis C, Fiziev P, Papp B, Butz S, Bonora G, Sabri S et al. Cooperative Binding of Transcription Factors Orchestrates Reprogramming. Cell 2017; 168: 442–59. e20. Michael AK, Thomä NH. Reading the chromatinized genome. Cell 2021; 184: 3599–3611. Larson ED, Marsh AJ, Harrison MM. Pioneering the developmental frontier. Mol Cell 2021; 81: 1640–50. Jiang J, Chan Y-S, Loh Y-H, Cai J, Tong G-Q, Lim C-A et al. A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat Cell Biol 2008; 10: 353–60. Soufi A, Donahue G, Zaret KS. Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome. Cell 2012; 151: 994–1004. Yolland L, Burki M, Marcotti S, Luchici A, Kenny FN, Davis JR et al. Persistent and polarized global actin flow is essential for directionality during cell migration. Nat Cell Biol 2019; 21: 1370–81. Schoenfelder S, Fraser P. Long-range enhancer-promoter contacts in gene expression control. Nat Rev Genet 2019; 20: 437–55. Winick-Ng W, Kukalev A, Harabula I, Zea-Redondo L, Szabó D, Meijer M et al. Cell-type specialization is encoded by specific chromatin topologies. Nature 2021; 599: 684–91. Goel VY, Huseyin MK, Hansen AS. Region Capture Micro-C reveals coalescence of enhancers and promoters into nested microcompartments. Nat Genet 2023; 55: 1048–56. Steeg PS. Targeting metastasis. Nat Rev Cancer 2016; 16: 201–18. Weiss F, Lauffenburger D, Friedl P. Towards targeting of shared mechanisms of cancer metastasis and therapy resistance. Nat Rev Cancer 2022; 22: 157–73. Bushweller JH. Targeting transcription factors in cancer - from undruggable to reality. Nat Rev Cancer 2019; 19: 611–24. Morita K, Suzuki K, Maeda S, Matsuo A, Mitsuda Y, Tokushige C et al. Genetic regulation of the RUNX transcription factor family has antitumor effects. J Clin Invest 2017; 127: 2815–28. Békés M, Langley DR, Crews CM. PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov 2022; 21: 181–200. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupplementaryMaterials.pdf SupplementaryTableS1.xlsx SupplementaryTableS2.xlsx Cite Share Download PDF Status: Published Journal Publication published 22 Apr, 2024 Read the published version in Oncogene → Version 1 posted Editorial decision: revise 20 Oct, 2023 Review # 1 received at journal 17 Oct, 2023 Review # 2 received at journal 06 Oct, 2023 Reviewer # 2 agreed at journal 03 Oct, 2023 Reviewer # 1 agreed at journal 29 Sep, 2023 Reviewers invited by journal 28 Sep, 2023 Submission checks completed at journal 27 Sep, 2023 Editor assigned by journal 26 Sep, 2023 First submitted to journal 26 Sep, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Wei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYFACxgZphgrStZwh1R5pxjZSlBscb268XTivLnH+jOSHHxgq7tk1sJ89gF/LmYPN1jO3HU7ccCPNWILhTHFyA09eAl4tZjcS26R5tx1I3CCRw8bA2JaQzCDBY4Bfy/2HQC1zQA4jWssNRqCWBubEhhsQLXYEtdifSWy25jl22HjDmWfGEglnEhLYeHLwa5FsP/7wNk9Nnez8dmCIfahIsOdnP4NfCww4NoDIBAaGRKLjyB6DMQpGwSgYBaMABgA9mkOn1f2kMQAAAABJRU5ErkJggg==","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":true,"prefix":"","firstName":"Denghui","middleName":"","lastName":"Wei","suffix":""},{"id":236786780,"identity":"d6ea95ac-0c79-4a3f-8632-b158c46aadff","order_by":1,"name":"Zhenyu Yang","email":"","orcid":"https://orcid.org/0000-0001-8442-1029","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Yang","suffix":""},{"id":236786781,"identity":"39f0ccf6-1d51-426b-9674-8e92d0563419","order_by":2,"name":"Yanfu Peng","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Yanfu","middleName":"","lastName":"Peng","suffix":""},{"id":236786782,"identity":"b4872074-8924-4199-8373-101a4eca7663","order_by":3,"name":"Yaqin Wang","email":"","orcid":"","institution":"State Key Laboratory of Oncology in Southern China, Collaborative Innovation Center of Cancer Medicine, Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Yaqin","middleName":"","lastName":"Wang","suffix":""},{"id":236786783,"identity":"3a08c988-f666-4432-a6ad-04a8e071a3a6","order_by":4,"name":"Panyang Yang","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Panyang","middleName":"","lastName":"Yang","suffix":""},{"id":236786784,"identity":"57a13cb7-88c6-46a7-912d-82ccc82af759","order_by":5,"name":"Zhuohui Huang","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Zhuohui","middleName":"","lastName":"Huang","suffix":""},{"id":236786785,"identity":"dc7944bb-46bc-4000-89ee-d85f35083a5e","order_by":6,"name":"Tingqiu Quan","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Tingqiu","middleName":"","lastName":"Quan","suffix":""},{"id":236786786,"identity":"b32587d0-1b48-41aa-9b27-abc8f71156a0","order_by":7,"name":"Xudong Xu","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Xudong","middleName":"","lastName":"Xu","suffix":""},{"id":236786787,"identity":"9bcb9417-1152-48f6-a72f-c64bae7394ae","order_by":8,"name":"Ying Sun","email":"","orcid":"https://orcid.org/0000-0002-5888-2929","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Sun","suffix":""},{"id":236786788,"identity":"098e160f-eedc-4ab6-ae2f-598038a8043d","order_by":9,"name":"Guan-Qun Zhou","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Guan-Qun","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2023-09-27 03:52:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3390645/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3390645/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41388-024-03033-0","type":"published","date":"2024-04-22T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55138854,"identity":"51c4437c-ebb2-48ff-8498-c80b788cfd51","added_by":"auto","created_at":"2024-04-23 07:16:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":496823,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3390645/v1/7ebabef2-795c-429e-9ae8-11de896f242b.pdf"},{"id":44145443,"identity":"3bac9a0e-f214-47d0-9904-d65e6fc05c32","added_by":"auto","created_at":"2023-10-05 15:26:17","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1756600,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3390645/v1/f13e696c22b95163b7964970.pdf"},{"id":44145444,"identity":"b97ebca9-ace5-4435-a36f-57ca2577fed3","added_by":"auto","created_at":"2023-10-05 15:26:18","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":12823359,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3390645/v1/9f3d6469a86eb2cf4ebdf53a.xlsx"},{"id":44147829,"identity":"1bbf83d6-7a33-4a4b-9cac-04d21894ebb5","added_by":"auto","created_at":"2023-10-05 15:42:17","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":105492,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3390645/v1/1763cbd78769866b18389203.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"KLF5 regulates actin remodeling to enhance the metastasis of nasopharyngeal carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMetastasis, the heterogeneous and systemic disease, compromises the function of distant organs colonized by aggressive cancer cells, causing disruption of homeostasis and eventually death [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Cancer metastasis is a cell motility-based multistep pathological cascade during which cancer cells disseminate from the primary tumor, hijack tumor microenvironment, intravasate and extravasate from vessels, enter and exit dormancy, accommodate to various tissue conditions and colonize distant organs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. During metastasis, orchestration of actin cytoskeleton dynamics renders cancer cells sensing and responding to physical stimuli to augment cell motility and dissemination [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Cells precisely reshape by actin remodeling to resist fluid shear stress in bodily circulatory systems, survive and seed metastatic lesions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs the basis of metastasis, cell migration encompasses a series of cellular events. After integrating and transmitting cell-intrinsic stimuli and environment-generated cues, metastatic cells initiate the polarization of actin cytoskeletal machinery at the cell front and subsequently form protrusions [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Protrusions at the leading edge driven by rearranged dense actin networks generate asymmetric or polarized membrane tension to direct cell migration [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Intriguingly, actin-rich nascent membrane protrusions extend from areas with low density of membrane-proximal F-actin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Lamellipodia, a flatter type of protrusion with fan-like architecture, is dominated by Arp2/3-branched actin, attributed to activation of nucleation-promoting factors by Rho-family GTPases [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Another mechanism of actin-driven migration shows that, the orchestrated retrograde actin flow creates retrograde shear forces, propelling cells to migrate following the environmental topography [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn response to promigratory signals, rapid transcription modulation by transcription factors (TFs) during cell migration facilitates adaption of cells facing changing extracellular milieu [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Human Kr\u0026uuml;ppel-like factors (KLFs) are a family of 17 transcription factors interacting with DNA through conserved triple C2H2 zinc fingers (ZnFs) domain [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. KLFs control various key cellular processes including differentiation, inflammation and migration, with exclusive and redundant functions of each KLF [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In cancers, derailed expression and function of KLFs under divergent contexts implicate cancer pathogenesis, heterogeneity, therapeutic resistance, recurrence and metastasis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. A recent study revealed the KLF4-mediated exquisite transcription modulation model involving enhancers [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the reprogramming of mouse embryonic fibroblasts, KLF4 reorganizes the chromatin structure and rewires three-dimensional enhancer loops, leading to adaptive transcriptional changes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. KLFs cross-regulate and co-opt in cancer development and malignant progression, albeit their intricate regulatory networks and effects remain to be fully determined.\u003c/p\u003e \u003cp\u003eNasopharyngeal carcinoma (NPC) is a nasopharynx epithelium originated carcinoma with a high metastatic proclivity and special geographic distribution that in 2020, \u0026gt;\u0026thinsp;75% new cases occurred in Eastern Asia and South-Eastern Asia, especially in China [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. After first-line induction chemotherapy plus concurrent chemoradiotherapy treatment, approximately 25% patients still developed recurrence or distant metastasis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For recurrent or metastatic NPC patients receiving first-line treatment, the median progression-free survival of 7.6 months was suboptimal [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the master TF involved in distant metastasis of NPC remains elusive.\u003c/p\u003e \u003cp\u003eIn this study, we discover that KLF5 operates as a master TF and preferentially occupies distal enhancer regions to regulate transcription output in metastatic NPC. We further show that KLF5 transcriptionally activates ACTN4 by binding to its conserved enhancer to facilitate actin remodeling, extending of actin-rich lamellipodia and distant metastasis of NPC.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eKLF5 is a master TF of metastatic NPC\u003c/h2\u003e \u003cp\u003eTo investigate the master TF engaged in distant metastasis of NPC, we first leveraged the Algorithm for the Reconstruction of Accurate Cellular Networks through Adaptive Partitioning (ARACNe-AP) to build transcriptional interactomes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] with a multitude of TF-target interplay derived from gene expression profile of curated datasets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Based on the interactomes across 1,639 known or likely TFs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], we generated the intricate TFs regulatory networks by Virtual Inference of Protein-activity by Enriched Regulon analysis based on multiple samples (msVIPER) algorithm to assess transcriptional activity of master TFs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and identified 192 candidates for distant metastasis of NPC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, 2). We delightedly found that three KLFs (KLF5, KLF7 and KLF3) in 192 active TFs presumably promotes NPC metastasis (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Compared to other KLFs, the expression of \u003cem\u003eKLF5\u003c/em\u003e ranked top in NPC samples and those with distant metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). Given its highest activity and expression among three active KLFs in metastatic samples of NPC (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-E), we speculated that KLF5 operates as a master TF and promotes distant metastasis of NPC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next analyzed two public datasets (GSE103611 and GSE13597) and identified KLF5\u003csup\u003ehigh\u003c/sup\u003e and KLF5\u003csup\u003elow\u003c/sup\u003e human NPC groups, and KLF5\u003csup\u003ehigh\u003c/sup\u003e group showed a larger proportion of patients with distant metastasis and stage III\u0026ndash;IV disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-G). Gene set enrichment analysis (GSEA) of the transcriptome of these two groups revealed a pronounced enrichment of the metastasis relevant gene set in KLF5\u003csup\u003ehigh\u003c/sup\u003e NPC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). These results indicate that NPC patients with higher expression and transcriptional activity of KLF5 are prone to develop distant metastasis and progress to an advanced stage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eKLF5 enhances metastatic potential of NPC cells in vitro and in vivo\u003c/h2\u003e \u003cp\u003eWe compared the expression level of \u003cem\u003eKLF5\u003c/em\u003e in immortalized N2-Tert nasopharyngeal mucosa cells and 6 NPC cell lines. The results showed that \u003cem\u003eKLF5\u003c/em\u003e expressed higher in NPC cells and samples than normal control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J and Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). Since KLF5 accelerated cell proliferation and malignant progression of pancreatic ductal adenocarcinoma [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], short hairpin RNA (shRNA)-mediated knockdown (KD) of \u003cem\u003eKLF5\u003c/em\u003e was used to investigate the role of KLF5 in the carcinogenesis of hyperproliferative NPC. Cell viability and colony formation results showed that, knockdown of \u003cem\u003eKLF5\u003c/em\u003e did not alter the proliferation kinetics of NPC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB-E). Therefore, we focused on the role of KLF5 in distant metastasis of NPC and the underlying mechanism. Compared with control cells, knockdown of \u003cem\u003eKLF5\u003c/em\u003e triggered an indolent metastatic phenotype that suppressing cell migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eF). Moreover, depletion of \u003cem\u003eKLF5\u003c/em\u003e significantly increased the time required for wound closure on 2D surfaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eG, H). Accordingly, overexpression of \u003cem\u003eKLF5\u003c/em\u003e potentiated the motility of NPC cells in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E and Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eI, J). We further constructed the lung metastatic model and inguinal lymph node metastatic model wherein \u003cem\u003eKLF5\u003c/em\u003e-KD or control NPC cells were injected into the tail vein or footpad of immunocompromised mice, respectively. Compared to the control, mice intravenously injected with \u003cem\u003eKLF5\u003c/em\u003e-KD NPC cells shouldered less lung metastatic burden showing reduction of overt metastatic lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Consistently, knockdown of \u003cem\u003eKLF5\u003c/em\u003e precluded metastatic dissemination of NPC cells from primary tumor in footpad to groin, leading to smaller tumor-draining lymph nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Knockdown of \u003cem\u003eKLF5\u003c/em\u003e constrained NPC cell infiltration of normal tissues including skin, muscle and lung in metastatic models (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, I). Collectively, our data suggests that KLF5 enhances migratory propensity of cells in vitro and metastatic outbreak in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eKLF5 regulates actin remodeling and lamellipodia formation\u003c/h2\u003e \u003cp\u003eNext, we sought to identify the molecular features and pathways determining KLF5-enhanced metastatic potential of NPC. Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) and Reactome pathway enrichment analysis of KLF5\u003csup\u003ehigh\u003c/sup\u003e and KLF5\u003csup\u003elow\u003c/sup\u003e human NPCs in different datasets revealed that the actin cytoskeleton reorganization, Rho-family GTPases and cell motility associated pathways are enriched in KLF5\u003csup\u003ehigh\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). We applied GESA analysis to compare the transcriptome of these two groups, and observed a pronounced enrichment of gene sets germane to actin cytoskeleton, wound healing, Rho-family GTPases and EMT in KLF5\u003csup\u003ehigh\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D and Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDiverse guidance cues converge into asymmetric or polarized traction force generation to direct cell migration, with leading-edge protrusions acting as one pivotal executor [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Most protrusions are driven by the continuous polymerization and depolymerization of branched and linear arrays of actin, of which lamellipodia appears flatter and is dominated by Arp2/3-branched actin [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. To evaluate whether KLF5 augments cell motility by tuning actin remodeling and lamellipodia formation, we first implemented RNA-sequencing (RNA-seq) analysis of \u003cem\u003eKLF5\u003c/em\u003e-KD and control NPC cells. By KEGG, GO and GSEA analysis on the transcriptome of \u003cem\u003eKLF5\u003c/em\u003e-KD and control cells, we identified enriched functional categories of actin cytoskeleton, focal adhesion, wound healing and cell migration regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F and Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD-F). Second, we conducted knockdown or overexpression of \u003cem\u003eKLF5\u003c/em\u003e in another individual assay to verify the transcriptomic changes occurring in \u003cem\u003eKLF5\u003c/em\u003e-depleted metastatic cells. Knockdown of \u003cem\u003eKLF5\u003c/em\u003e inhibited expression of many key genes in the pathway of actin cytoskeleton regulation, whereas overexpression of \u003cem\u003eKLF5\u003c/em\u003e elicited contrast effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H and Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eG, H). Aligned with in silico analysis, depletion of \u003cem\u003eKLF5\u003c/em\u003e interrupted the dynamics of actin networks and induced unstructured actin polymerization, attenuated lamellipodia growth and unpolarized cell shapes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-K). Accordingly, control cells plated on 2D surfaces generated denser and more branched cortical actin filament arrays in cytoplasm and lamellipodia at the leading edge, forming larger F-actin-rich lamellipodia and increasing cell spreading area (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-K). Taken together, these results demonstrate that KLF5-dependent actin branching drives lamellipodia growth and cell spreading, enhancing the cell motility in vitro.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eKLF5 preferentially occupies distal enhancer regions\u003c/h2\u003e \u003cp\u003eGiven the canonical role of KLF5 in interpreting the genome, we employed calibrated chromatin immunoprecipitation sequencing (ChIP-seq) analysis to estimate the sequence preference and genomic binding loci of KLF5. To depict KLF5-related epigenetic landscape of NPC cells, we mined genome-wide profiles of NPC cell assessed by ChIP-seq (GSE95749) and analyzed distinct signal distribution pattern of epigenetic marks in the vicinity of KLF5 peaks. Based on signal of chromatin marks in C666-1 cells, the regions of KLF5 peaks were divided into three clusters: promoter (10.7%), enhancer (74.6%) and other regions (without marks, 14.7%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Clusters of KLF5 peaks displayed similar distribution in HK1 cells (Supplementary Fig. S4A). Most KLF5-occupied promoter and enhancer regions extensively overlapped with accessible regions inferred by assay for transposase-accessible chromatin (ATAC), suggestive of their accessibility to transcription factors and related collaborators (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B and Supplementary Fig. S4B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong grouped KLF5 peaks, H3K4me1 indicated enhancer, H3K4me3 indicated promoter and H3K27ac indicated enhancer and promoter in all regions, respectively (Supplementary Fig. S4C-E). In H3K27ac\u003csup\u003e+\u003c/sup\u003e regions, the H3K4me3:H3K27ac ratio was higher than H3K4me1:H3K27ac in promoter cluster, whereas enhancer cluster exhibited contrast manifestation and other undefined regions exhibited no significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). While location of KLF5 peaks annotated to specified genomic regions, we observed similar effect that H3K4me1:H3K27ac was higher in intergenic regions and gene body, and H3K4me3:H3K27ac was higher in promoters (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Approximately 15% of KLF5 peaks were located within compacted chromatin lacking ATAC signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). The metagene analysis showed that H3K27ac, H3K4me1 and H3K4me3 modifications exhibited bimodal distribution pattern around KLF5 peak summit in enhancer or promoter cluster, while ATAC signal exhibited unimodal distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Supplementary Fig. S4A). These results suggested that KLF5 occupancy may propel nucleosome removal or further DNA unwrapping, rendering relevant DNA surfaces accessible to KLF5 and other DNA-binding proteins.\u003c/p\u003e \u003cp\u003eTo examine occupancy preference of KLF5 at \u003cem\u003ecis\u003c/em\u003e-regulatory elements and genomic regions, we grouped KLF5 peaks into quartiles based on the peak height and analyzed the distribution of KLF5 peaks relative to annotated genes. Overall, only 22% of KLF5 peaks were situated in proximity to (\u0026plusmn;\u0026thinsp;1kb) transcriptional start site (TSS), whereas 47% of KLF5 peaks were further than 10 kb to the closest TSS, and the proportion of peaks adjacent to TSS was inversely proportional to KLF5 affinity to genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Most (74.6%) KLF5 peaks occurred in enhancers that probably most were distal enhancers while KLF5 weakly bound most (73.3%) promoters (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Consistently, KLF5 preferred occupying distal regions (intergenic and gene body regions) instead of promoters, and the preference increased with the strength of KLF5 binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G and Supplementary Fig. S4F). To further characterize the KLF5 binding pattern in genome, we grouped KLF5 peaks into four fractions depending on H3K4me1:H3K27ac and H3K4me3:H3K27ac ratio. Among four quadrants, quadrant II (H3K4me1:H3K27ac+, H3K4me3:H3K27ac-) contained largest proportion of enhancers (32%) and distal regions (33%), while quadrant IV (H3K4me1:H3K27ac-, H3K4me3:H3K27ac+) contained most promoters (57%), supporting the notion that H3K4me1 marks enhancers and H3K4me3 marks promoters (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and Supplementary Fig. S4G). Quadrant IV contained most (27%) Q1 KLF5 peaks, quadrant II contained most (29\u0026ndash;30%) Q2 and Q3 KLF5 peaks, and quadrant I contained most (69%) Q4 KLF5 peaks, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Taken together, these findings uncover the previously undetermined pattern that KLF5 preferentially occupies the distal enhancer regions rather than promoters to regulate transcription.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKLF5 transcriptionally activates\u003c/b\u003e \u003cb\u003eACTN4\u003c/b\u003e \u003cb\u003eby occupying its enhancer\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify the prominent target transcriptionally activated by KLF5 during cell migration, we analyzed the biological function of KLF5 \u003cem\u003ecis\u003c/em\u003e-acting targets inferred by ChIP-seq through GO and KEGG pathway enrichment analysis, and found these genes implicated actin cytoskeleton regulation, lamellipodia formation and cell migration, which was consistent with analysis on KLF5-grouped transcriptome of NPC cells and samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-F). Among candidates involved in actin cytoskeleton regulation (ACTN4, ITGB5, VCL, VAV2, BAIAP2), alpha-actinin 4 (ACTN4), a protein engaging with dynamics of actin filaments [\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], proficiently recruited KLF5 to its gene body (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). With visualization of ChIP-seq data of KLF5 and histone modifications and ATAC-seq data in NPC cells on UCSC genome browser, we observed that, in \u003cem\u003eACTN4\u003c/em\u003e gene body, enhancer marks (H3K4me1 and H3K27ac) and ATAC signal was enriched in the KLF5-binding region with relatively low H3K4me3 signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and Supplementary Fig. S5A). Notably, KLF5 ChIP-seq for cells with various inherent characteristics uncovered similar binding peaks in \u003cem\u003eACTN4\u003c/em\u003e gene body (Supplementary Fig. S5B), ranging from squamous cell carcinoma (SCC), gastric cancer (GC), colorectal cancer (CRC) to pancreatic ductal adenocarcinoma (PDAC). Simultaneously, the KLF5-binding region showed similar chromatin marks distribution and DNase I hypersensitivity in diverse cell types (Supplementary Fig. S5C), indicating this region as a conserved \u003cem\u003eACTN4\u003c/em\u003e enhancer captured by KLF5 and recruitment of KLF5 to \u003cem\u003eACTN4\u003c/em\u003e enhancer is a pervasive and heretofore undefined phenomenon.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the dominant KLF5 binding loci in the \u003cem\u003eACTN4\u003c/em\u003e enhancer, we discovered top six consensus motifs enriched in KLF5 peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Supplementary Fig. S5D). Since \u003cem\u003eACTN4\u003c/em\u003e enhancer contained two potential motifs for KLF5 binding, we hypothesized that KLF5 binds \u003cem\u003eACTN4\u003c/em\u003e enhancer at these two motifs and transcriptionally activates \u003cem\u003eACTN4\u003c/em\u003e. To address it, we first generated KLF5 protein models by multiple tools, of which the best model predicted by Phyre2 was used for subsequent HDOCK molecular docking [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In KLF5-motif docking models (confidence score: 0.91, 0.86), ZnFs of KLF5 contacted the DNA major groove by hydrogen bond interactions between specific residues and bases (Supplementary Fig. S6A-C). In KLF5-motif1 model, residues Y384, S387, K391 and T396 of ZnF1 and W412 of ZnF2 contacted bases C2, G5\u0026rsquo;, G7\u0026rsquo;, C12, respectively (Supplementary Fig. S6B). In KLF5-motif2 model, residues T396 of ZnF1 and S445 and R454 of ZnF3 contacted bases G1\u0026rsquo;, G6\u0026rsquo;, C3, respectively (Supplementary Fig. S6C). The specific residues contacting \u003cem\u003eACTN4\u003c/em\u003e enhancer overlapped with a transcriptional activation domain of KLF5 identified recently [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] (Supplementary Fig. S6A). Given the high docking confidence, we confirmed that endogenous KLF5 bound the two motifs in \u003cem\u003eACTN4\u003c/em\u003e enhancer and another motif in \u003cem\u003eACTN4\u003c/em\u003e promoter as evaluated by ChIP-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F and Supplementary Fig. S6D). The two motifs in \u003cem\u003eACTN4\u003c/em\u003e enhancer were marked by H3K27ac as well (Supplementary Fig. S6E-G). For orthogonal enhancer activity assays, we generated a luciferase reporter incorporating \u003cem\u003eACTN4\u003c/em\u003e promoter and reporters incorporating two tandem gene cassettes: \u003cem\u003eACTN4\u003c/em\u003e promoter followed by full-length or truncated \u003cem\u003eACTN4\u003c/em\u003e enhancer with one motif. Compared to pGL3-Basic reporter, introduction of \u003cem\u003eACTN4\u003c/em\u003e regulatory sequences substantially activated luciferase transcription, with strongest activation by full-length \u003cem\u003eACTN4\u003c/em\u003e enhancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H and Supplementary Fig. S6H, I). Exogenous KLF5 further enhanced the luciferase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H and Supplementary Fig. S6H, I). We next mutated both two motifs and found diminished luciferase activity upon the mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI and Supplementary Fig. S6H, I), indicating that KLF5-bound loci dominate transcriptional activation ability of the \u003cem\u003eACTN4\u003c/em\u003e enhancer. Analysis of Hi-C data of human umbilical vein endothelial cells (HUVEC) in a 3D-genome Interaction Viewer and database [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] showed the direct interplay between \u003cem\u003eACTN4\u003c/em\u003e enhancer and \u003cem\u003eACTN4\u003c/em\u003e promoter (Supplementary Fig. S7). These results together demonstrate that KLF5 occupies \u003cem\u003eACTN4\u003c/em\u003e enhancer and facilitates \u003cem\u003eACTN4\u003c/em\u003e enhancer-promoter interplay to activate \u003cem\u003eACTN4\u003c/em\u003e transcription, potentially through chromatin looping or compartmentalization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eACTN4-actin conjunction enhances lamellipodia formation and cell motility\u003c/h2\u003e \u003cp\u003eSince KLF5 transcriptionally activated \u003cem\u003eACTN4\u003c/em\u003e in NPC cells, the positive correlation between \u003cem\u003eKLF5\u003c/em\u003e and \u003cem\u003eACTN4\u003c/em\u003e expression was confirmed in NPC samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B and Supplementary Fig. S8A, B). \u003cem\u003eACTN4\u003c/em\u003e was upregulated in NPC samples, analogous to \u003cem\u003eKLF5\u003c/em\u003e expression pattern (Supplementary Fig. S8C). As opposed to ACTN4\u003csup\u003elow\u003c/sup\u003e group, ACTN4\u003csup\u003ehigh\u003c/sup\u003e group matched higher potential for metastatic progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and Supplementary Fig. S8D). GSEA analysis revealed the metastasis relevant gene set enrichment in ACTN4\u003csup\u003ehigh\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), suggesting that ACTN4 engages metastatic pathways in NPC patients. We next knocked down \u003cem\u003eACTN4\u003c/em\u003e in NPC cells, which mimicked \u003cem\u003eKLF5\u003c/em\u003e-silenced metastatic phenotypes of inferior capacity to migrate and invade in short-term migration and wound healing assays in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-H and Supplementary Fig. S8E-G), indicating that ACTN4 imparts enhanced metastatic potential to NPC cells. Accordingly, overexpression of \u003cem\u003eACTN4\u003c/em\u003e conveyed augmented metastatic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI, J and Supplementary Fig. S8H-J). Introduction of ACTN4 partially rescued the impaired metastatic activity of \u003cem\u003eKLF5\u003c/em\u003e-KD NPC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK, L and Supplementary Fig. S8K, L).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn view of our data showing that KLF5 regulated actin remodeling and lamellipodia formation, as well as the well-established role of ACTN4 in actin dynamics and cell motility [\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], we speculated that ACTN4, transcriptionally activated by KLF5, crosslinks with actin filaments, triggering denser lamellipodial actin networks and lamellipodia growth. We next applied GSEA analysis of the expression profiles from ACTN4\u003csup\u003ehigh\u003c/sup\u003e and ACTN4\u003csup\u003elow\u003c/sup\u003e NPC groups, which revealed an active state of actin networks and lamellipodia organization at the cell leading edge underlying faster cell migration in ACTN4\u003csup\u003ehigh\u003c/sup\u003e NPC group (Supplementary Fig. S8M). We further performed immunofluorescence analysis to detect the distribution of ACTN4 and actin filaments in \u003cem\u003eACTN4-\u003c/em\u003eoverexpressed NPC cells. ACTN4 colocalized with F-actin but not with dissociative actin in the whole cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Intriguingly, exogenous ACTN4 induced accumulation of actin filaments in lamellipodia rather than in the whole cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). Moreover, larger area of lamellipodia were detected in cells with exogenous ACTN4, enhancing cell spread on 2D surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). Radial line profile analysis showed that, F-actin intensively localized throughout the extending lamellipodia at the cell edge, and ACTN4 displayed similar distribution pattern that the fluorescence intensity increased with distance from nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). In line with previous study [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], exogenous ACTN4 substantially colocalized with highly branched and dense actin filament networks, especially at the cell leading edge and membrane lamellipodia (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Colocalization analysis corroborated the extensive colocalization between ACTN4 and F-actin in HONE-1 (Pearson\u0026rsquo;s correlation value: 0.8; Manders\u0026rsquo; colocalization coefficients: tM1\u0026thinsp;=\u0026thinsp;0.908 and tM2\u0026thinsp;=\u0026thinsp;0.901) and SUNE-1 cells (Pearson\u0026rsquo;s correlation value: 0.7; Manders\u0026rsquo; colocalization coefficients: tM1\u0026thinsp;=\u0026thinsp;0.836 and tM2\u0026thinsp;=\u0026thinsp;0.819). Knockdown of \u003cem\u003eACTN4\u003c/em\u003e impaired the ACTN4-actin conjunction and formation of branched actin networks, causing defective cell morphology and lamellipodia extending (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Collectively, these results demonstrate the critical role of ACTN4 in remodeling actin networks at the cell edge to facilitate lamellipodia formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we elucidate that KLF5 is a master TF that preferentially occupies the \u003cem\u003eACTN4\u003c/em\u003e enhancer to activate its transcription, which regulates the formation of actin-driven lamellipodia and enhances the intrinsic metastatic potential of NPC cells in vitro and in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Through analyzing transcriptional interactome and regulatory network, we identified KLF5 as a master TF governing transcription program in NPC metastasis and exacerbation. Emerging evidence reveals that KLF5 promotes cancer metastasis under diverse contexts through divergent pathways [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Our analysis suggests that NPC patients bearing high KLF5 burden are prone to develop distant metastasis and progress to an advanced stage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBy systematically analyzing the KLF5-related epigenetic landscape assessed by ChIP-seq and ATAC-seq, we discover the previously undetermined phenomenon that a large group of KLF5 peaks (74.6%) occur at distal regions coinciding with enhancer \u003cem\u003ecis\u003c/em\u003e-regulatory elements in NPC, indicating its occupancy preference for distal enhancer regions. Enrichment of H3K27ac and ATAC signal in KLF5-captured promoters and enhancer regions reveals the chromatin accessibility and transcription active state of these regions associated genes. Accumulating evidence has linked KLF4 with dynamic enhancer organization which directs spatiotemporal gene expression programs in pluripotent stem cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Previous chromatin profiling studies showed that KLF4 cooperating with reprogramming factors mainly occupies enhancers in reprogramming and established pluripotent stem cells [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. KLF4 orchestrates the long-range chromatin loops rendering physical contacts between enhancers and promoters through interaction with architectural proteins such as cohesion [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Recruitment of pioneer factors to chromatinized binding sites opens and remodels the chromatin and facilitates subsequent binding of other TFs and cofactors [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. KLF4 is a pioneer factor promoting reprogramming and the impaired pluripotency due to loss of KLF4 can be compensated by other KLFs (e.g., KLF5), indicating potential pioneer factor identity of them [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Given the bimodal distribution of histone marks and unimodal distribution of ATAC signal around KLF5 peak summit, whether KLF5 operates as a pioneer factor and reads out nucleosomal DNA in compacted chromatin to facilitate appropriate nucleosome occupancy and position needs further study.\u003c/p\u003e \u003cp\u003eOrchestrated by transcription modulation, cell migration predominantly depends on precise tuning of global actin flow at the cell front and rear [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Our analysis shows that KLF5 interprets the genome to respond to promigratory cues, triggering rapid modulation of genes involved in actin cytoskeleton transcriptionally. Among these candidates, \u003cem\u003eACTN4\u003c/em\u003e proficiently recruits KLF5 to its gene body. Integrating ChIP-seq data of chromatin marks and KLF5 under various contexts, we discovered a heretofore unrecognized and conserved enhancer element in \u003cem\u003eACTN4\u003c/em\u003e gene body occupied by KLF5. Intriguingly, introducing truncated \u003cem\u003eACTN4\u003c/em\u003e enhancer with one motif slightly impaired the transcriptional activation strength of \u003cem\u003eACTN4\u003c/em\u003e promoter with or without ectopic KLF5, presumably attributed to sequence integrity requirement for stabilizing the enhancer-promoter interplay. Alternatively, the truncated \u003cem\u003eACTN4\u003c/em\u003e enhancer lacks potential to recruit sufficient KLF5 and co-binding transcription activators capturing flanking sequences, hence some repressive TFs and corepressors partially substitute for KLF5. Given canonical regulatory modes of long-range enhancer-promoter crosstalk [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], KLF5 on \u003cem\u003eACTN4\u003c/em\u003e enhancer may recruit coactivators and promote the chromatin loop extrusion or genome compartmentalization to synergistically drive transcription. While the structure of KLF5-DNA complex remains undetermined, we simulated the docking of putative KLF5 structure and its binding motif in \u003cem\u003eACTN4\u003c/em\u003e enhancer inferred by ChIP-seq and found a high confidence of the KLF5-motif docking model. Additional essential biochemical information will be obtained by high-resolution structure research of purified KLF5 and its partners. Recent comprehensive measurements of human transcriptional effector domains revealed that KLF5 boasts activation and repression domains, rendering the bifunctional potential of KLF5 in specific contexts [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Thus, in response to distinct cues, KLF5 potentially oscillates between activating and repressing activity at various genomic loci and the regulatory output can be delineated by stoichiometries.\u003c/p\u003e \u003cp\u003eThe transcription activation of \u003cem\u003eACTN4\u003c/em\u003e by KLF5 explains the impaired actin network structure, actin-rich lamellipodia, cell spreading and directed migration caused by depletion of KLF5 in NPC cells. ACTN4-actin conjunction empowers metastatic NPC cells to generate actin-driven lamellipodia and efficiently migrate faster to seek congenial milieu to survive and grow. Though studying KLF5-ACTN4 facilitated cell migration using 2D systems reveals the mechanism of how KLF5 enhances the metastatic potential of cells in vitro and in vivo, utilization of tools and more complex systems in more physiological contexts can deepen our understanding of migration principles underlying metastasis, through which providing promising therapeutic avenues to control metastasis \u0026mdash; the overwhelming cause of cancer-associated death.\u003c/p\u003e \u003cp\u003eTo prevent metastasis and eliminate established metastatic lesions, numerous agents have been developed to identify and drug targets in metastatic cascade, of which TFs are switching from \u0026lsquo;undruggable\u0026rsquo; to \u0026lsquo;druggable\u0026rsquo; [\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. However, clinical validation of TF-targeting agents and incorporating them into the standard of care remains problematic, owing to their cytostatic manifestation in preclinical models and compensatory pathways involving requisite steps in metastasis [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Therefore, rational combination therapies are efficacious in targeting metastasis. KLF5, the dysregulated master TF in metastatic NPC, represents a unique potential therapeutic target controlling metastasis, with challenges of developing inhibitors targeting its protein-DNA and protein-protein interplay [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Though inhibitors targeting KLF5-DNA binding remain to be developed, a recent effort overcame the difficulty of inhibitor recognizing convex and highly positively charged DNA binding interfaces [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. An agent targeting RUNX-DNA binding increased survival of mouse xenograft models [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Inhibitors interrupting binding of KLF5 on \u003cem\u003eACTN4\u003c/em\u003e enhancer and other loci may serve as means to mitigate metastases of NPC. Moreover, utilizing the induced proximity principle and ubiquitin-proteasome system, the proteolysis-targeting chimaera molecules potentially degrade KLF5 through a ligand binding KLF5 and another ligand recruiting ubiquitin ligases [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, our discoveries underscore the prometastatic role of KLF5, a master TF in NPC, and provide mechanistic insights to identify potential biomarkers and therapeutic interventions to shrink metastases. Novel agents targeting mutated or dysregulated KLF5 and other master TFs in combination with first-line therapies will improve outcome of patients with metastatic disease.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptional interactome and regulatory network inference\u003c/h2\u003e \u003cp\u003eThe transcriptional interactomes were generated by ARACNe-AP algorithm [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] from 4 GEO datasets profiled by RNA-seq or microarray: GSE118719, GSE68799, GSE13597 and GSE103611. ARACNe-AP was run with 100 reproducible bootstrap iterations using retrieved gene expression profiles and 1,639 TFs as predefined input, with parameters setting to no DPI (Data Processing Inequality) tolerance and MI (Mutual Information) \u003cem\u003ep\u003c/em\u003e-value threshold of 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e. Inferred significant KLF-target interactions were filtered (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for subsequent analysis. With input of transcriptional interactomes and gene expression profiles, regulatory networks were reverse engineered to output regulons.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTFs activity inference\u003c/h2\u003e \u003cp\u003eTFs activity profiles were estimated by msVIPER algorithm [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], which tests regulon enrichment on gene expression signatures. By comparing NPC samples under specific contexts (e.g., metastatic versus non-metastatic), gene expression signatures were generated to identify potential role of TFs in NPC progression. After randomly permuted samples of gene expression profiles 1,000 times, null models were produced by using signatures generated with permutation iterations. Comparing each regulon enrichment score to a null model, TFs activity was inferred as normalized enrichment score, facilitating the identification of master TFs in distant metastasis of NPC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell cultrure\u003c/h2\u003e \u003cp\u003eSix human NPC cell lines (C666-1, CNE1, CNE2, HNE-1, HONE-1 and SUNE-1) were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; ExCell Bio). Human immortalized normal nasopharyngeal epithelial cell line (N2-Tert) was maintained in keratinocyte serum\u0026ndash;free medium (Invitrogen), supplemented with bovine pituitary extract (BD Biosciences). The above cell lines were authenticated and generously provided by M.S. Zeng (SYSUCC, China). Human Embryonic Kidney 293T (HEK293T) cells obtained from the ATCC was maintained in DMEM (Gibco) supplemented with 10% FBS (ExCell Bio).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, RT-PCR and qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells using either TRIzol reagent (Invitrogen) or RNA Quick Purification kit (ESscience) according to the manufacturer\u0026rsquo;s instructions, following RNA quality and quantity measurement by NanoDrop 2000 (Thermo Fisher Scientific). cDNA was generated using HiScript III RT SuperMix for qPCR (Vazyme) according to the manufacturer\u0026rsquo;s instructions. cDNA was diluted 1:10 in distilled water and 2 \u0026micro;l was used per qPCR reaction.\u003c/p\u003e \u003cp\u003eqRT-PCR was performed using SYBR qPCR Master Mix (Vazyme) on CFX96 Touch sequence detection system (Bio-Rad). Primers were designed using the Primer-BLAST tool at NCBI. Housekeeping gene mRNA level (GAPDH) was used for normalization. The mRNA levels of all genes were quantified using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method to infer the difference. qPCR primer sets are listed in Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eCells were lysed on ice using the radioimmunoprecipitation assay (RIPA, Merck Millipore) supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland) for 30 min and centrifuge at 4\u0026deg;C for 15 min at 12,000 rpm. The protein concentrations of supernatants were determined using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Total protein lysates (30 \u0026micro;g) were separated by SDS\u0026ndash;polyacrylamide gel electrophoresis (4\u0026ndash;20%) and transferred to PVDF membranes (Merck Millipore). Membranes were blocked for 1 h in 5% skimmed milk and then incubated overnight at 4\u0026deg;C in primary antibody solution. After washing in TBST, membranes were incubated with secondary antibodies for 1h at room temperature. Proteins were detected using Super ECL Plus (Applygen). The following primary antibodies were used: anti-KLF5 (rabbit; Sigma-Aldrich, 09822, 1:2000), anti-HA (rabbit; Abcam, ab9110, 1:10000), anti-ACTN4 (rabbit; Abcam, ab108198, 1:5000), anti-GAPDH (rabbit; Abcam, ab181602, 1:10000), anti-alpha Tubulin (mouse; Abcam, ab7291, 1:10000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTransient transfection and stable cell line construction\u003c/h2\u003e \u003cp\u003eFor siRNA-mediated RNA interference, siRNAs targeting human \u003cem\u003eKLF5\u003c/em\u003e and non-targeting control were synthesized and ordered from GenePharma (Suzhou, China). Human \u003cem\u003eKLF5\u003c/em\u003e or \u003cem\u003eACTN4\u003c/em\u003e CDS region cDNA tagged by HA was cloned into the pSin-EF2-Puro plasmid (Addgene) using BamHI and EcoRI restriction sites. The siRNAs or plasmids were transfected to cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer\u0026rsquo;s instructions. The shRNAs targeting human \u003cem\u003eKLF5\u003c/em\u003e and \u003cem\u003eACTN4\u003c/em\u003e or non-targeting control were cloned into the pLKO.1-Puro plasmid (Addgene) using AgeI and EcoRI restriction sites. Lentiviral particles were generated by transfecting pLKO.1-shRNA or pSin-EF2-cDNA and psPAX2 plasmid (Addgene) and pMD2G plasmid (Addgene) in HEK293T cells with polyethyleneimine (PEI; Polysciences). Lentiviral supernatants were harveseted 48 h after transfection and were filtered by a 0.45 \u0026micro;m pore size PVDF membrane (jet). HONE-1 and SUNE-1 cells were infected by lentiviruses with polybrene (Beyotime). After 24 h infection, stable cell lines were selected by puromycin for 72 h and verified by western blotting. A list of targeted sequences for siRNA and shRNA is provided in Supplementary Table S4 and 5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCell viability and colony formation assays\u003c/h2\u003e \u003cp\u003eFor the cell viability assay, 1,000 cells were seeded to each well of 96-well plates (jet) in 10% FBS-added medium and cultured for 5 days. After incubated for 12 h, cell viability was measured by a CCK-8 kit (APExBIO) according to the manufacturer\u0026rsquo;s instructions and measured every 24 hours after first measurement. Absorbance at 450 nm was recorded by a spectrophotometric plate reader (BioTek ELX800, Bio-Rad). For colony formation assay, 400 cells were seeded to each well of six-well plates (jet) in 10% FBS-added medium and cultured for 10\u0026ndash;14 days. Detectable colonies were fixed with methanol for 10 min, stained with hematoxylin for 1 h and analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assay\u003c/h2\u003e \u003cp\u003eCells were seeded into six-well plates (jet) and cultured to a 100% confluency, following a scratch using a 200 \u0026micro;l pipette tip and a plastic ruler as a guide in the cell monolayers. Detached cells were washed by phosphate-buffered saline (PBS), after which images were captured by the inverted microscope (NIKON Eclipse Ti2-U). Remaining cells were allowed for 24 h growing and were imaged with the inverted microscope (NIKON Eclipse Ti2-U). Images were analysed using Fiji/ImageJ by drawing a line indicating the migrating fronts on two sides of the scratch wound and comparing healing rate between groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTranswell migration and invasion assays\u003c/h2\u003e \u003cp\u003eFor Transwell migration and invasion assays, 8 \u0026micro;m pore size transwell chambers (corning) were coated with or without Matrigel (BD Biosciences) according to the manufacturer\u0026rsquo;s instructions. Cells resuspended in 200 \u0026micro;l serum-free medium were seeded into the upper chambers with 3.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e HONE-1 cells or 4.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e SUNE-1 cells for migration assay and 7 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e HONE-1 cells or 9 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e SUNE-1 cells for invasion assay. Lower chambers were filled with 500 \u0026micro;l 10% FBS-added medium. After incubation for 12\u0026ndash;14 hours (migration assay) or 20\u0026ndash;22 hours (invasion assay), the migrated or invaded cells were fixed with methanol for 10 min, stained with hematoxylin for 2 h and imaged by inverted microscope (NIKON Eclipse Ti2-U). Cell migration and invasion capability were defined as relative number of migrated or invaded cells manually counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo metastasis models\u003c/h2\u003e \u003cp\u003e In vivo metastasis experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Ethics Committee of SYSUCC (L025504202207003). BALB/c nude mice (female, 4\u0026ndash;6 weeks old, 15g) were obtained from Charles River Laboratories (Beijing, China) and maintained at the Animal Experiment Center of the Sun Yat-sen University.\u003c/p\u003e \u003cp\u003eFor the lung metastasis model, \u003cem\u003eKLF5\u003c/em\u003e-KD or control HONE-1 cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) in PBS were injected into the tail vein of mice (n\u0026thinsp;=\u0026thinsp;6 per group). One month after injection, mice were euthanized and their lungs were dissected and analyzed for metastasis.\u003c/p\u003e \u003cp\u003eFor the inguinal lymph node metastasis model, \u003cem\u003eKLF5\u003c/em\u003e-KD or control HONE-1 and SUNE-1 cells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) in PBS were inoculated into the footpads of mice (n\u0026thinsp;=\u0026thinsp;6 per group and n\u0026thinsp;=\u0026thinsp;10 per group). One month after inoculation, mice were euthanized and their footpad primary tumors and inguinal tumor-draining lymph nodes were dissected and analyzed for metastasis.\u003c/p\u003e \u003cp\u003eThe primary tumors and lungs were fixed in 4% formaldehyde for 48 h. Tissues were embedded in paraffin, sectioned into 5 \u0026micro;m pieces, and mounted on slides for hematoxylin and eosin staining. Slides were imaged using an Olympus scanning system (VS200).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence and image analysis\u003c/h2\u003e \u003cp\u003eFor Immunofluorescence assay, cells were fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100 and blocked in 3% BSA for 1h at room temperature. Cells were incubated at 4\u0026deg;C with an anti-HA antibody (rabbit; ab9110; 1:1000) diluted in 1% BSA solution overnight. After washing three times with PBS, cells were incubated for 1 h at room temperature with Alexa Fluor 594 goat anti-rabbit IgG H\u0026amp;L (abcam; ab150080; 1:500). diluted in 1% BSA solution. Nuclei and F-actin were then stained with DAPI (Biosharp) and phalloidin-Alexa Fluor 488 (Beyotime), respectively. After washing three times with PBS, cells were mounted using antifade mounting medium (Beyotime). Cells were imaged using a 63\u0026times; oil-immersion objective on a Zeiss confocal system (LSM 980).\u003c/p\u003e \u003cp\u003eMean intensity of F-actin and spreading area in the whole cell and lamellipodia were recorded through manually drawing the free-form regions of interest (ROI) using Fiji/ImageJ. For radial line profile analysis, line scans of captured images were generated through lamellipodial outlines and the cell edge was applied as a reference to evaluate the spatial distribution of ACTN4 and F-actin. The Pearson correlation coefficient (r) was calculated between the mean intensity of F-actin and ACTN4 at different distance. For colocalization analysis, images of cells expressing ectopic ACTN4 were exported to Fiji and colocalization efficiency and specificity of ACTN4 and F-actin was analyzed using the Fiji Coloc2 plugin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eChIP\u003c/h2\u003e \u003cp\u003eChIP assay was performed using Pierce Magnetic ChIP Kit (26157, Thermo Fisher Scientific) following the manufacturer\u0026rsquo;s instructions. Briefly, 4 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cultured HONE-1 or SUNE-1 cells were harvested and washed with ice-cold PBS twice. Cells were fixed in 2ml of 1% formaldehyde for 10 min at room temperature, and Glycine Solution (10\u0026times;) was added to a final concentration of 1\u0026times; to quench crosslink for 5 min at room temperature, following two washes with ice-cold PBS. Cells were collected by mechanically scrapping in 1mL of ice-cold PBS with 10 \u0026micro;L of the Halt Cocktail and centrifuged at 3,000 \u0026times; g for 5 min at 4\u0026deg;C. After removing PBS, crosslinked cells were lysed using Membrane Extraction Buffer containing protease/phosphatase inhibitors and digested using MNase. After sonication on ice to break nuclear membrane, solutions were centrifuged at 9,000 \u0026times; g for 5 min at 4\u0026deg;C. 10% of the supernatants containing the digested chromatin was stored as input sample, and the remaining supernatants were incubated with primary antibodies: 1 \u0026micro;L IgG and 3\u0026ndash;5 \u0026micro;g anti-KLF5 (rabbit; Sigma-Aldrich, 09822), anti-HA (rabbit; Abcam, ab9110) or anti-H3K27ac (rabbit; ab177178) at 4\u0026deg;C overnight with mixing. Protein A/G Magnetic Beads were added and incubated for 2 hours at 4\u0026deg;C with mixing. Beads were collected with a magnetic stand and were washed three times with IP Wash Buffer 1 and once with IP Wash Buffer 2. The protein-DNA complexes were eluted from beads and de-crosslinked using IP Elution Buffer containing NaCl and Proteinase K for 3 hours at 65\u0026deg;C. DNA samples were then purified and subjected to qPCR or ChIP-seq library generation. Primers were provided in Supplementary Table S6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase reporter assay\u003c/h2\u003e \u003cp\u003ePromoters of \u003cem\u003eACTN4\u003c/em\u003e (1,000 bp upstream of the transcription start site) and truncated \u003cem\u003eACTN4\u003c/em\u003e enhancer containing motif1/2 or full-length \u003cem\u003eACTN4\u003c/em\u003e enhancer or mutant \u003cem\u003eACTN4\u003c/em\u003e enhancer (all bases of motifs replaced with adenosines) were cloned into pGL3-basic luciferase reporter plasmid (Promega). Inserted sequences were provided in Supplementary Table S7. Cells were seeded into 24-well plates and co-transfected with plasmids encoding an empty vector or HA-tagged \u003cem\u003eKLF5\u003c/em\u003e (400 ng) and luciferase reporter plasmids (100 ng) and a Renilla luciferase reporter (Addgene, 10 ng) using Lipofectamine 3000 (Invitrogen). Cells were lysed after 24-48h of transfection by Dual-Luciferase Reporter Assay System (E1910, Promega) following the manufacturer\u0026rsquo;s instructions. Luciferase activity was measured by GloMax Navigator (Promega), and firefly luciferase activity was normalized to Renilla luciferase activity. Luciferase mRNA level in different groups were evaluated through RNA extraction, RT-PCR and qPCR.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eChIP-seq\u003c/h2\u003e \u003cp\u003eThe ChIP-seq libraries were prepared using Paired-End DNA Sample Prep kit (Illumina) according to the manufacturer\u0026rsquo;s instructions. The libraries were amplified by PCR with 18 cycles and size selected for 100-300bp fragments. Qualified libraries were sequenced on a NextSeq 500 Sequencer (Illumina) according to the manufacturer\u0026rsquo;s instructions. Raw reads were filtered and quality-verified using SOAPnuke program (v.2.0). After filtering, the clean data was mapped to human genome GRCh38 by SOAP2 (v.2.21). Peaks were called using model-based MACS2 software (v.2.1.1) and annotated with the closest hg38 genes by annotatePeaks of HOMER (v.4.11). Comparing peaks from two ChIP-seq libraries (IgG and KLF5 groups), KLF5-unique peaks were identified by MAnorm (v.1.2.0), which met the standard that |M| \u0026gt;= 1 and \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. The ChIP-seq data for this study are available for download from the Gene Expression Omnibus (GEO) repository (GSE243951).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis and visualization of ChIP-seq, ATAC-seq and Hi-C data\u003c/h2\u003e \u003cp\u003eThe following ChIP-seq and ATAC-seq data were downloaded from the publicly available datasets: GSE95749 (NPC), including C666-1_H3K4me1, C666-1_H3K4me3, C666-1_H3K27ac, C666-1_ATAC, HK1_H3K4me1, HK1_H3K4me3 and HK1_H3K27ac; GSE88976 (SCC), BICR31_KLF5; GSE51705 (GC), KATOIII_KLF5; GSE49402 (CRC), LoVo_KLF5; GSE64557 (PDAC), CFPAC-1_KLF5. The genome reference of public ChIP-seq and ATAC-seq data were converted from human hg19 to hg38 using CrossMap (v.0.5.4). ChIP-seq and ATAC-seq identified peaks and normalized signal on gene tracks were visualized using UCSC Genome Browser. KLF5 and chromatin marks occupancy on genomic regions were created using the computeMatrix and the plotHeatmap tools of deepTools (v.3.5.2). Based on signal of chromatin marks, KLF5 ChIP-seq regions were categorized into three clusters: promoter regions (H3K27ac and H3K3me3-enriched), enhancer regions (H3K27ac and H3K3me1-enriched) and other undefined regions. Metaplots revealing KLF5 and chromatin marks enrichment in different regions were created using the plotProfile tool of deepTools (v.3.5.2). ChIP-seq and ATAC-seq identified peaks were annotated to specified genomic regions (promoter, peaks occurring at \u0026plusmn;\u0026thinsp;1kb intervals around the hg38 gene transcription start site; gene body, peaks occurring at gene bodies; intergenic, peaks occurring outside promoters and gene bodies) using ChIPseeker (v.1.28.3). Analysis of a Hi-C contact map in HUVEC cell was performed using a 3D-genome Interaction Viewer and database (3DIV) to generate chromatin interactions between \u003cem\u003eACTN4\u003c/em\u003e promoter and \u003cem\u003eACTN4\u003c/em\u003e enhancer.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eMotif enrichment analysis\u003c/h2\u003e \u003cp\u003eFor motif discovery, the motifs in KLF5 peaks of ChIP-seq were discovered using MEME (v.4.10.1) with the significance cutoff E value\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e. For motif comparison, the discovered motifs in KLF5 peaks of ChIP-seq were compared to known motifs in HOCOMOCOv11 database using Tomtom (v.5.5.3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eFor RNA-seq analysis of HONE-1 cells with or without knockdown of \u003cem\u003eKLF5\u003c/em\u003e, total RNA was extracted from cells using TRIzol reagent (Invitrogen). Purified mRNA using Dynabeads Oligo (dT) (Thermo Fisher) was fragmented by NEBNext Magnesium RNA fragmentation module (NEB) and reverse-transcribed to create the cDNA by SuperScript\u0026trade; II Reverse Transcriptase (Invitrogen), following U-labeled second-stranded DNAs synthesis with E. coli DNA polymerase I (NEB), RNase H (NEB) and dUTP Solution (Thermo Fisher). The final cDNA libraries were constructed by random hexamer primed cDNA synthesis, PCR amplification and size selection as 300\u0026thinsp;\u0026plusmn;\u0026thinsp;50 bp. The cDNA libraries were sequenced on the Illumina Novaseq\u0026trade; 6000 platform according to the manufacturer\u0026rsquo;s instructions. Raw reads were filtered and quality-verified using cutadapt (v.1.9.1) and FastQC (v.0.11.9) before alignment. Reads were aligned and assembled using HISAT2 (v.2.2.1) and StringTie (v.2.1.6) against the human genome GRCh38. Transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare software (v.0.9.8). Transcript-level and gene-level were quantified using StringTie and ballgown. Normalization of raw count data and genes differential expression analysis were performed using the DESeq2 software. The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 were considered differentially expressed genes (DEGs), subjected to following GO, KEGG and GSEA analysis. The RNA-seq data for this study are available for download from the Gene Expression Omnibus (GEO) repository (GSE243952).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eGO and KEGG analysis\u003c/h2\u003e \u003cp\u003eGO and KEGG enrichment analysis were performed against Human MSigDB (v2023.1. Hs) collections c2 (KEGG subset of Canonical pathways) and c5 (Gene Ontology gene sets) for genes ranging from DEGs from RNA-seq of HONE-1 cells with or without knockdown of \u003cem\u003eKLF5\u003c/em\u003e, DEGs identified from KLF5\u003csup\u003ehi\u003c/sup\u003e and KLF5\u003csup\u003elo\u003c/sup\u003e groups (categorized by KLF5 median expression level) in GSE13597 and GSE103611 datasets to KLF5 \u003cem\u003ecis\u003c/em\u003e-acting candidates in ChIP-seq.\u0026nbsp;GO terms and pathways meeting this condition with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were defined as significantly enriched GO terms and pathways in DEGs.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eGene set enrichment analysis (GSEA)\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eGene set enrichment analysis was performed using GSEA (v4.1.0) with default parameters against Human MSigDB (v2023.1. Hs) collections c2 (KEGG subset and Reactome subset of Canonical pathways), c5 (Gene Ontology gene sets), metastasis-associated gene sets and hallmark gene sets. Differentially enriched gene sets were analyzed for \u003cem\u003eKLF5\u003c/em\u003e-KD and control RNA-seq dataset and GEO datasets which were grouped into KLF5\u003csup\u003ehi\u003c/sup\u003e and KLF5\u003csup\u003elo\u003c/sup\u003e groups or ACTN4\u003csup\u003ehi\u003c/sup\u003e and ACTN4\u003csup\u003elo\u003c/sup\u003e groups. |NES|\u0026gt;1, NOM \u003cem\u003ep\u003c/em\u003e-val\u0026thinsp;\u0026lt;\u0026thinsp;0.05, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.25 were considered to be different in two groups.\u003c/p\u003e \u003cp\u003e \u003cb\u003eModel generation of KLF5 and molecular docking simulation of KLF5-\u003c/b\u003e \u003cb\u003eACTN4\u003c/b\u003e \u003cb\u003eenhancer motif complex\u003c/b\u003e\u003c/p\u003e \u003cp\u003eKLF5 protein models were generated using five specific systems based on homology modeling method: SWISS-MODEL, Phyre2, I-Tasser, Robetta and AlphaFold2 and one system based on Ab initio prediction method: trRosetta. The quality of models for each system were assessed by their own scoring functions and other assessment programs including ProQ, Verify3D, PROCHECK and SolVX for three criteria: spatial geometry quality, stereochemistry quality and energetical stability. The best model predicted by Phyre2 was used for subsequent HDOCK molecular docking with the two motifs in \u003cem\u003eACTN4\u003c/em\u003e enhancer, respectively. The docking models with best docking score and confidence score were selected to perform molecular graphical analysis using UCSF ChimeraX (v.1.6.1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed by GraphPad Prism 9 (GraphPad Software). \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant; *, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. For data with Gaussian distributions, statistical comparison was made using Two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test between two groups and one-way ANOVA for more than two groups. Data represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD unless stated otherwise.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article and its Supplementary Information Files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China\u0026nbsp;(92259202, 82273401, 81872463), the Guangdong Basic and Applied Basic Research Foundation (2019A1515011863,\u0026nbsp;2019A1515012045, 2023B1515020014),\u0026nbsp;the Health \u0026amp; Medical Collaborative Innovation Project of Guangzhou City, China (201803040003) and the Special Support Program of Sun Yat-sen University Cancer Center (16zxtzlc06).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGZ and DW designed and supervised the study. GZ, DW and ZY conducted the literature search. ZY, YP, YW and PY performed the experiments. ZY and XX performed the bioinformatic analysis. ZH developed the animal models. TQ, ZY and YP extracted and analyzed data. GZ, ZY, DW and YS were responsible for interpreting results. ZY, YW, DW and GZ contributed to the figures and tables. ZY and DW wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGerstberger S, Jiang Q, Ganesh K. Metastasis. Cell 2023; 186: 1564\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMassagu\u0026eacute; J, Ganesh K. Metastasis-Initiating Cells and Ecosystems. Cancer Discov 2021; 11: 971\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFollain G, Herrmann D, Harlepp S, Hyenne V, Osmani N, Warren SC et al. Fluids and their mechanics in tumour transit: shaping metastasis. Nat Rev Cancer 2020; 20: 107\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSenGupta S, Parent CA, Bear JE. The principles of directed cell migration. Nat Rev Mol Cell Biol 2021; 22: 529\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu A, Yao B, Dong T, Chen Y, Yao J, Liu Y et al. Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer. Cell 2022; 185: 1356\u0026ndash;72. e26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBera K, Kiepas A, Godet I, Li Y, Mehta P, Ifemembi B et al. Extracellular fluid viscosity enhances cell migration and cancer dissemination. Nature 2022; 611: 365\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHakala M, Wioland H, Tolonen M, Kotila T, Jegou A, Romet-Lemonne G et al. Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks. Nat Cell Biol 2021; 23: 147\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanerjee T, Biswas D, Pal DS, Miao Y, Iglesias PA, Devreotes PN. Spatiotemporal dynamics of membrane surface charge regulates cell polarity and migration. Nat Cell Biol 2022; 24: 1499\u0026ndash;1515.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReversat A, Gaertner F, Merrin J, Stopp J, Tasciyan S, Aguilera J et al. Cellular locomotion using environmental topography. Nature 2020; 582: 582\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMehidi A, Kage F, Karatas Z, Cercy M, Schaks M, Polesskaya A et al. Forces generated by lamellipodial actin filament elongation regulate the WAVE complex during cell migration. Nat Cell Biol 2021; 23: 1148\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Belly H, Yan S, Borja da Rocha H, Ichbiah S, Town JP, Zager PJ et al. Cell protrusions and contractions generate long-range membrane tension propagation. Cell 2023; 186: 3049\u0026ndash;61. e15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBisaria A, Hayer A, Garbett D, Cohen D, Meyer T. Membrane-proximal F-actin restricts local membrane protrusions and directs cell migration. Science 2020; 368: 1205\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLappalainen P, Kotila T, J\u0026eacute;gou A, Romet-Lemonne G. Biochemical and mechanical regulation of actin dynamics. Nat Rev Mol Cell Biol 2022; 23: 836\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Nie L, Zhang Y, Yan Y, Wang C, Colic M et al. Actin cytoskeleton vulnerability to disulfide stress mediates disulfidptosis. Nat Cell Biol 2023; 25: 404\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou H, Poore B, Brown EE, Qian J, Xie B, Asimakidou E et al. A neurodevelopmental epigenetic programme mediated by SMARCD3-DAB1-Reelin signalling is hijacked to promote medulloblastoma metastasis. Nat Cell Biol 2023; 25: 493\u0026ndash;507.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTetreault M-P, Yang Y, Katz JP. Kr\u0026uuml;ppel-like factors in cancer. Nat Rev Cancer 2013; 13: 701\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, L\u0026uuml;ttmann FF, Schoger E, Sch\u0026ouml;ler HR, Zelaray\u0026aacute;n LC, Kim K-P et al. Reversible reprogramming of cardiomyocytes to a fetal state drives heart regeneration in mice. Science 2021; 373: 1537\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlacher E, Tsai C, Litichevskiy L, Shipony Z, Iweka CA, Schneider KM et al. Aging disrupts circadian gene regulation and function in macrophages. Nat Immunol 2022; 23: 229\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Yang L, Wu P, Li X, Tang Y, Ou X et al. The circROBO1/KLF5/FUS feedback loop regulates the liver metastasis of breast cancer by inhibiting the selective autophagy of afadin. Mol Cancer 2022; 21: 9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng L, Zhu Y, Moreno CS, Wan Y. New insights into KLFs and SOXs in cancer pathogenesis, stemness, and therapy. Semin Cancer Biol 2023; 90: 29\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Giammartino DC, Kloetgen A, Polyzos A, Liu Y, Kim D, Murphy D et al. KLF4 is involved in the organization and regulation of pluripotency-associated three-dimensional enhancer networks. Nat Cell Biol 2019; 21: 1179\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\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. CA Cancer J Clin 2021; 71: 209\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerlay J, Ervik M, Lam F, Colombet M, Mery L, Pi\u0026ntilde;eros M, Znaor A, Soerjomataram I, Bray F. Global Cancer Observatory: Cancer Today. Lyon, France: International Agency for Research on Cancer. 2020. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gco.iarc.fr/today\u003c/span\u003e\u003cspan address=\"https://gco.iarc.fr/today\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 2 Aug 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Chen L, Hu G-Q, Zhang N, Zhu X-D, Yang K-Y et al. Gemcitabine and Cisplatin Induction Chemotherapy in Nasopharyngeal Carcinoma. N Engl J Med 2019; 381: 1124\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Pan J, Wang H, Zhao Y, Qu S, Chen N et al. Tislelizumab plus chemotherapy as first-line treatment for recurrent or metastatic nasopharyngeal cancer: A multicenter phase 3 trial (RATIONALE-309). Cancer Cell 2023; 41: 1061\u0026ndash;72. e4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLachmann A, Giorgi FM, Lopez G, Califano A. ARACNe-AP: gene network reverse engineering through adaptive partitioning inference of mutual information. Bioinformatics 2016; 32: 2233\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLambert SA, Jolma A, Campitelli LF, Das PK, Yin Y, Albu M et al. The Human Transcription Factors. Cell 2018; 172: 650\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlvarez MJ, Shen Y, Giorgi FM, Lachmann A, Ding BB, Ye BH et al. Functional characterization of somatic mutations in cancer using network-based inference of protein activity. Nat Genet 2016; 48: 838\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe P, Yang JW, Yang VW, Bialkowska AB. Kr\u0026uuml;ppel-like Factor 5, Increased in Pancreatic Ductal Adenocarcinoma, Promotes Proliferation, Acinar-to-Ductal Metaplasia, Pancreatic Intraepithelial Neoplasia, and Tumor Growth in Mice. Gastroenterology 2018; 154: 1494\u0026ndash;1508.e13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMueller J, Szep G, Nemethova M, de Vries I, Lieber AD, Winkler C et al. Load Adaptation of Lamellipodial Actin Networks. Cell 2017; 171: 188\u0026ndash;200.e16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaplan JM, Kim SH, North KN, Rennke H, Correia LA, Tong HQ et al. Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis. Nat Genet 2000; 24: 251\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang M-C, Chang Y-H, Wu C-C, Tyan Y-C, Chang H-C, Goan Y-G et al. Alpha-actinin 4 is associated with cancer cell motility and is a potential biomarker in non-small cell lung cancer. J Thorac Oncol 2015; 10: 286\u0026ndash;301.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Z, Zhou J-K, Wang K, Chen H, Qin S, Liu J et al. PDLIM1 Inhibits Tumor Metastasis Through Activating Hippo Signaling in Hepatocellular Carcinoma. Hepatology 2020; 71: 1643\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng D, Kumar M, Muntel J, Gurley SB, Birrane G, Stillman IE et al. Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis. J Am Soc Nephrol 2020; 31: 1479\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 2015; 10: 845\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan Y, Tao H, He J, Huang S-Y. The HDOCK server for integrated protein-protein docking. Nat Protoc 2020; 15: 1829\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelRosso N, Tycko J, Suzuki P, Andrews C, Aradhana, Mukund A et al. Large-scale mapping and mutagenesis of human transcriptional effector domains. Nature 2023; 616: 365\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim K, Jang I, Kim M, Choi J, Kim M-S, Lee B et al. 3DIV update for 2021: a comprehensive resource of 3D genome and 3D cancer genome. Nucleic Acids Res 2021; 49: D38\u0026ndash;D46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang B, Li Y, Wu Q, Xie L, Barwick B, Fu C et al. Acetylation of KLF5 maintains EMT and tumorigenicity to cause chemoresistant bone metastasis in prostate cancer. Nat Commun 2021; 12: 1714.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu P, Wang Z, Ou X, Wu P, Zhang Y, Wu S et al. The FUS/circEZH2/KLF5/ feedback loop contributes to CXCR4-induced liver metastasis of breast cancer by enhancing epithelial-mesenchymal transition. Mol Cancer 2022; 21: 198.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Q, Liu M, Zhang D, Lin B-B, Fu X, Zhang Z et al. Nitazoxanide inhibits acetylated KLF5-induced bone metastasis by modulating KLF5 function in prostate cancer. BMC Med 2023; 21: 68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei Z, Gao F, Kim S, Yang H, Lyu J, An W et al. Klf4 organizes long-range chromosomal interactions with the oct4 locus in reprogramming and pluripotency. Cell Stem Cell 2013; 13: 36\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChronis C, Fiziev P, Papp B, Butz S, Bonora G, Sabri S et al. Cooperative Binding of Transcription Factors Orchestrates Reprogramming. Cell 2017; 168: 442\u0026ndash;59. e20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichael AK, Thom\u0026auml; NH. Reading the chromatinized genome. Cell 2021; 184: 3599\u0026ndash;3611.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarson ED, Marsh AJ, Harrison MM. Pioneering the developmental frontier. Mol Cell 2021; 81: 1640\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang J, Chan Y-S, Loh Y-H, Cai J, Tong G-Q, Lim C-A et al. A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat Cell Biol 2008; 10: 353\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoufi A, Donahue G, Zaret KS. Facilitators and impediments of the pluripotency reprogramming factors\u0026rsquo; initial engagement with the genome. Cell 2012; 151: 994\u0026ndash;1004.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYolland L, Burki M, Marcotti S, Luchici A, Kenny FN, Davis JR et al. Persistent and polarized global actin flow is essential for directionality during cell migration. Nat Cell Biol 2019; 21: 1370\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchoenfelder S, Fraser P. Long-range enhancer-promoter contacts in gene expression control. Nat Rev Genet 2019; 20: 437\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinick-Ng W, Kukalev A, Harabula I, Zea-Redondo L, Szab\u0026oacute; D, Meijer M et al. Cell-type specialization is encoded by specific chromatin topologies. Nature 2021; 599: 684\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoel VY, Huseyin MK, Hansen AS. Region Capture Micro-C reveals coalescence of enhancers and promoters into nested microcompartments. Nat Genet 2023; 55: 1048\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteeg PS. Targeting metastasis. Nat Rev Cancer 2016; 16: 201\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeiss F, Lauffenburger D, Friedl P. Towards targeting of shared mechanisms of cancer metastasis and therapy resistance. Nat Rev Cancer 2022; 22: 157\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBushweller JH. Targeting transcription factors in cancer - from undruggable to reality. Nat Rev Cancer 2019; 19: 611\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorita K, Suzuki K, Maeda S, Matsuo A, Mitsuda Y, Tokushige C et al. Genetic regulation of the RUNX transcription factor family has antitumor effects. J Clin Invest 2017; 127: 2815\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB\u0026eacute;k\u0026eacute;s M, Langley DR, Crews CM. PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov 2022; 21: 181\u0026ndash;200.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3390645/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3390645/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTranscription factors (TFs) engage in various cellular essential processes including differentiation, growth and migration. However, the master TF involved in distant metastasis of nasopharyngeal carcinoma (NPC) remains largely unclear. Here we show that KLF5 regulates actin remodeling to enhance NPC metastasis. We analyzed the msVIPER algorithm-generated transcriptional regulatory networks and identified KLF5 as a master TF of metastatic NPC linked to poor clinical outcomes. KLF5 regulates actin remodeling and lamellipodia formation to promote the metastasis of NPC cells in vitro and in vivo. Mechanistically, KLF5 preferentially occupies distal enhancer regions of \u003cem\u003eACTN4\u003c/em\u003e to activate its transcription, whereby decoding the informative DNA sequences. ACTN4, extensively localized within actin cytoskeleton, facilitates dense and branched actin networks and lamellipodia formation at the cell leading edge, empowering cells to migrate faster. Collectively, our findings reveal that KLF5 controls robust transcription program of \u003cem\u003eACTN4\u003c/em\u003e to modulate actin remodeling and augment cell motility which enhances NPC metastasis, and provide new potential biomarkers and therapeutic interventions for NPC.\u003c/p\u003e","manuscriptTitle":"KLF5 regulates actin remodeling to enhance the metastasis of nasopharyngeal carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-05 15:26:12","doi":"10.21203/rs.3.rs-3390645/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2023-10-20T13:49:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-10-17T16:20:12+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-10-07T03:42:25+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-10-04T02:37:22+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-09-29T04:24:52+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2023-09-29T01:56:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-09-27T10:09:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-09-27T03:47:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogene","date":"2023-09-27T03:47:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4dbd970e-227d-41ee-a37f-b29f4dc7d92e","owner":[],"postedDate":"October 5th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":25072679,"name":"Biological sciences/Cancer/Metastasis"},{"id":25072680,"name":"Biological sciences/Molecular biology/Transcription"},{"id":25072681,"name":"Biological sciences/Cell biology/Cell migration"},{"id":25072682,"name":"Biological sciences/Genetics/Gene regulation"},{"id":25072683,"name":"Biological sciences/Cell biology/Cytoskeleton/Actin"}],"tags":[],"updatedAt":"2024-04-23T07:16:05+00:00","versionOfRecord":{"articleIdentity":"rs-3390645","link":"https://doi.org/10.1038/s41388-024-03033-0","journal":{"identity":"oncogene","isVorOnly":false,"title":"Oncogene"},"publishedOn":"2024-04-22 04:00:00","publishedOnDateReadable":"April 22nd, 2024"},"versionCreatedAt":"2023-10-05 15:26:12","video":"","vorDoi":"10.1038/s41388-024-03033-0","vorDoiUrl":"https://doi.org/10.1038/s41388-024-03033-0","workflowStages":[]},"version":"v1","identity":"rs-3390645","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3390645","identity":"rs-3390645","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}