Biology subjects

Tian, B.

Publications and source records attributed to Tian, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Single-cell lineage tracing maps clonal and transcriptional dynamics in melanoma metastasis

Melanoma metastasis is driven by extensive intratumoral heterogeneity and phenotypic plasticity, yet how clonal identity relates to transcriptional programs during metastasis remains unclear. Here, we applied MeRLin, a single-cell lineage tracing platform, to dissect the clonal and transcriptional heterogeneity of metastatic melanoma in a patient-derived spontaneous metastasis model. Clonal analyses revealed hierarchical structures during tumor progression, with a subset of lineages from primary tumors consistently enriched across metastatic sites, supporting a model of polyclonal seeding followed by selective expansion of pre-existing highly metastatic subpopulations. Single-cell transcriptomic profiling identified two major metastatic subpopulations of distinct transcriptional programs, characterized by neural crest stem cell-like and lipid metabolism signatures. Both programs were enriched for invasion-associated genes and maintained across organs through distinct regulatory networks. Spatial mapping by barcode RNA-FISH linked these transcriptional states to their tissue context and showed that OLFML3 expression partially co-localized with a dominant subpopulation at the tumor-liver interface, marking the invasive fronts of metastatic growth. Together, these findings establish a framework in which clonal identity, transcriptional state, and spatial organization jointly shape metastatic melanoma progression.

cancer biology↗

bioRxiv2026

Targeting CPSF73, the mRNA 3' End Processing Endonuclease, Moves Cancer Cells Away from the Mesenchymal State

BackgroundMetastasis significantly contributes to cancer-related mortality and therapeutic failure. Cancer cells acquire metastatic potential by losing epithelial characteristics and gaining mesenchymal properties through the epithelial-mesenchymal transition (EMT). Differential poly(A) site (PAS) usage, known as alternative polyadenylation (APA), generates mRNA isoforms differing in coding sequence, subcellular localization, stability, or translation efficiency. In cancer, 3'UTR shortening increases expression of proto-oncogenes by escaping miRNA-mediated repression. High expression of CPSF73, which cleaves mRNA precursors at PASs, is associated with unfavorable prognoses in cancer patients. However, the role of APA in regulating EMT remains poorly understood. MethodsIn this study, to investigate the role of APA in EMT, we employed JTE-607, a small-molecule inhibitor of CPSF73 activity, to examine the impact of catalytic inhibition of CPSF73 on proliferation and EMT in MDA-MB-231, MCF7, A549, and HepG2 cancer cells. To identify differential usage of PASs, global profiling of APA changes, and differential gene expression analysis were performed in MDA-MB-231 cells. Additionally, antisense oligonucleotides were used to block the use of a specific PAS whose APA change may be a driver of EMT reversal. ResultsOur findings showed that catalytic inhibition of CPSF73 not only attenuates cancer cell proliferation but also moves the cells away from the mesenchymal state across all four cell lines tested. Global profiling of APA changes following CPSF73 inhibition revealed widespread 3'UTR lengthening and suppression of intronic PASs in MDA-MB-231 cells. APA shifts were observed in key EMT-related genes, accompanied by decreased expression of corresponding proteins across all four cell lines. We used antisense morpholino oligonucleotides to block the proximal PAS of AKT2, shifting the balance of AKT2 mRNA isoforms toward the long isoform. This shift caused EMT reversal, marked by reduced AKT2 protein expression, changes in EMT-related markers, and impaired invasion by MDA-MB-231 cells. ConclusionTogether, these findings identify APA-mediated 3UTR lengthening, with functional consequences in EMT-related genes, as a coordinated mechanism leading to an attenuated EMT phenotype, highlighting a significant connection between APA and the EMT process. Interfering with these APA changes may offer a promising therapeutic strategy to suppress metastasis, with potential efficacy across multiple pathways. Statement of SignificanceOur findings highlight APA-mediated 3 UTR lengthening as a coordinated mechanism that promotes EMT reversal and support CPSF73 inhibition or APA-targeting strategies as potential therapeutic approaches to suppress metastasis across multiple pathways.

cancer biology↗

bioRxiv2026

High-Affinity Protein Binder Design via Flow Matching and In Silico Maturation

The de novo design of high-affinity protein binders remains a central challenge in protein engineering and therapeutic discovery. While deep generative models have advanced backbone generation and interface design, achieving picomolar or nanomolar binding affinities typically demands extensive experimental screening or iterative in vitro maturation. A general computational approach for the direct production of high-affinity binders has remained elusive. Here, we introduce an integrated framework that synergizes PPIFlow, a flow-matching-based generative model, with a novel in silico maturation strategy. PPIFlow employs a pairformer architecture to explicitly reason over pair-wise geometric and chemical interactions, modeling protein backbone rigid-body transformations as continuous flows. To bridge the affinity gap, we implement a dedicated in silico affinity maturation stage that combines interface rotamer enrichment with partial flow refinement to optimize energetic packing. This pipeline is further accelerated by AF3Score, a score-only adaptation of AlphaFold3 that enables high-fidelity and computationally efficient candidate prioritization. Across a diverse set of therapeutic targets, this synergistic approach consistently produces picomolar and nanomolar affinity binders without experimental affinity maturation. Notably, the framework proves highly effective for the de novo design of single-domain antibodies (VHHs), producing sub-nanomolar binders across multiple targets. These results establish that coupling robust backbone generation with focused in silico maturation renders the purely computational design of high-affinity binders feasible.

biochemistry↗

bioRxiv2025

ICFinder: ion channel identification and ion permeation residue prediction using protein language models

Ion channel dysfunction underlies many diseases (e.g., arrhythmias, epilepsy, cystic fibrosis), and uncharacterized channels may also contribute to pathology. Identifying such channels and their residues directly contacting the permeation pathway (i.e., ion permeation residues) is key to elucidating transport mechanisms and developing targeted therapies. Leveraging the protein language model ESM-2 and curated datasets, we developed BLAPE and CLAPE frameworks for high-throughput ion channel identification and permeation residue prediction. Our models outperformed existing methods, with 33%-171% improvements in Matthews correlation coefficients (MCC) across different datasets. Analysis of amino acid composition revealed enrichment for weakly polar residues among ion permeation sites. Case studies on four diverse ion channels highlighted that CLAPE consistently outperforms existing predictors and remains applicable to proteins lacking experimental structures, while also complementing structure-based pipelines such as AlphaFold3. We further applied our models to UniRef50 to predict potential ion channels, and made these results publicly available through the ICFinder webserver (https://tianlab-tsinghua.cn/icfinder/), providing a ready-to-use resource for the research community. All source code is available at https://github.com/JueWangTHU/ICFinder.

bioinformatics↗

bioRxiv2025

CodonTranslator: a conditional codon language model for codon optimization across all domains of life

Codon optimization involves selecting synonymous codons to match host-specific preferences. It is critical for heterologous expression but remains challenging due to the combinatorial design space. Under long-term evolutionary selection, natural coding sequences are near-optimal compromises between translational efficiency, accuracy, and regulatory constraints, providing a de facto standard for data-driven models. Recent deep learning-based language models therefore aim to learn the distribution of natural codon sequences and reuse it for design. However, existing approaches discard the rich semantic structure of taxonomic lineages, underutilize protein functional and evolutionary constraints, and often rely on masked-language objectives that lack a principled mechanism for sequence generation. Here we present CodonTranslator, a 150M-parameter decoder-only Transformer trained on 62 million CDS-protein pairs from over 2,100 species. CodonTranslator uses a pretrained language model to embed hierarchical species lineages and a pretrained protein language model to encode protein context, enabling interpolation across hosts and generalization to unseen species and proteins. Our results show that CodonTranslator implicitly learns the genetic code from data, faithfully reproduces species-specific codon usage, and designs coding sequences that match or surpass existing methods in both codon usage metrics and predicted biological stability. Our dataset, pretrained models, and code are available at https://github.com/poseidonchan/CodonTranslator

bioinformatics↗

bioRxiv2025

A disease mutation reveals a non-swapped pore and rewired gating cycle of the mechanically activated PIEZO1 channel

PIEZO1 is a versatile mechanotranduction channel critical for diverse cellular and physiological processes1-3. Gain-of-function mutations, such as R2482H, which slow channel inactivation, are linked to dehydrated hereditary stomatocytosis (DHS) and iron overload4-7. PIEZO1 forms a gigantic bowl-shaped trimer comprising flattenable peripheral blades and a central pore module with a switchable cap8-12. However, the structural dynamics governing its gating process and pathogenesis remain incompletely understood. Here, we determined distinct conformational states of the DHS-associated PIEZO1-R2482H mutant in both detergents and proteoliposomes. Remarkably, the R2482H mutation in the central pore induces a conformational change that propagates [~]25 nm to the most distal blade, enabling resolution of all 38 transmembrane helices. PIEZO1-R2482H is inherently flatter than PIEZO1, lowering the gating force and energy barrier and thus explaining its heightened mechanosensitivity. While the flattened PIEZO1 has its cap in the up-state adopting an inactivated state, the flattened PIEZO1-R2482H has its cap in the down-state and twisted by [~]75{degrees}, drastically switching the pore module from a swapped to a non-swapped organization. Such twisted conformation of PIEZO1-R2482H might hinder the transition to the inactivated state, instead proceeding through a deactivation-transition state characterized by curved blades and a partially twisted cap, thereby accounting for the slowed inactivation and deactivation kinetics. Taken together, we reveal the full transmembrane topology and a non-swapped pore conformation induced by R2482H, and propose the structural dynamic model that delineates the distinct gating cycle of activation, inactivation, and deactivation for PIEZO1 and PIEZO1-R2482H.

biophysics↗

bioRxiv2025

Bioengineered 3D hPSC-cholangiocyte ducts with physiological signals for biliary disease modelling

The progression of intrahepatic biliary diseases remains poorly understood, underscoring the urgent need to develop physiologically relevant human intrahepatic cholangiocyte disease models. Current approaches lack the complexity and throughput to capture how diverse biliary microenvironmental signals shape cholangiocyte behavior. To address this gap, we created a fully epithelialized and perfusible 3D bile duct from human pluripotent stem cell-derived cholangiocytes characterized by robust primary ciliation, apical-basal polarity and CFTR-mediated chloride conductance. Our results revealed physiologically relevant fluid flow and biliary stroma cells as essential components for sustaining cholangiocyte epithelial barrier integrity and ciliation. From here, we interrogated a broad spectrum of biliary signals to study bile acid toxicity and cytokine-driven injury, offering an unprecedented view into intrahepatic cholangiocyte stress responses and potential pathological mechanisms. By integrating physiologically relevant signals, fidelity and functional resolution, this platform provides the foundation needed to precisely decode pathogenic drivers and accelerate therapeutic development for devastating cholangiopathies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/686855v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@11c1255org.highwire.dtl.DTLVardef@1f78d11org.highwire.dtl.DTLVardef@14e9398org.highwire.dtl.DTLVardef@1bc08d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

HGS+ enlarged vesicular compartment serves as site for coronavirus assembly at later infection stage

The precise site of coronavirus assembly remains poorly characterized. In this study, we observed that coronavirus infection induces the host factor hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) to form distinct HGS enlarged vesicular compartments at later infection stage. Confocal and live-cell super-resolution microscopy showed that viral structural proteins colocalize with these HGS enlarged vesicular compartments. Correspondingly, APEX2-based electron microscopy (APEX2-EM) and immuno-EM analyses confirmed the presence of assembled virions within these unique HGS compartments, identifying them as sites of virion assembly. This was further supported by cryogenic correlated light and electron microscopy (cryo-CLEM), which captured ongoing virion formation occurring within HGS enlarged vesicles. Crucially, whole cell volume EM revealed that HGS deficiency abolishes these vesicular compartments and markedly reduces assembled virions. Lastly, we demonstrated that HGS vesicular compartments are rearranged from Golgi and endosome/lysosome by coronavirus infection. Together, these findings establish that coronavirus-induced HGS vesicular compartments function as essential platforms for virion assembly at later infection stage.

microbiology↗

bioRxiv2025

Targeting the host factor HGS-viral membrane protein interaction in coronavirus infection

While current antivirals primarily target viral proteins, host-directed strategies remain underexplored. Here, we performed a genome-wide CRISPRi screening to identify the host protein, Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate (HGS), as essential for the pan-coronaviruses infection both in vitro and in vivo. Mechanistically, HGS directly interacts with the viral membrane (M) protein, facilitating its trafficking to the ER-Golgi intermediate compartment (ERGIC) for virion assembly. Conversely, HGS deficiency caused M retention in the ER, blocking assembly. Leveraging this interaction, we designed M-derived peptides and screened over 5,000 FDA-approved drugs, identifying riboflavin tetrabutyrate (RTB). Both the peptides and RTB bind HGS and disrupt its interaction with the M protein, leading to M retention in the ER and subsequent blockade of virion assembly. These agents demonstrated broad anti-pan-coronavirus activity in vitro and in vivo. Collectively, our findings establish HGS as a druggable host target and identify RTB as a promising broad-spectrum antiviral candidate.

microbiology↗

bioRxiv2025

Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

Scalable generation of functionally mature hepatocytes from human pluripotent stem cells (hPSCs) is vital for regenerative medicine, disease modeling, and drug screening. We present a modular differentiation platform that enables serial expansion and cryopreservation of hepatic progenitors (hepatoblasts; HBs), supporting efficient downstream maturation. By modulating bile acid signaling with FGF19 and culturing under hypoxia, we preserved HB proliferation and bipotency. Subsequent thyroid hormone treatment induced alpha-fetoprotein-negative, albumin-positive hepatocytes, while WNT pathway modulation promoted zonal identity, mimicking in vivo hepatic metabolic heterogeneity. Zonation was confirmed by differential drug-metabolizing enzyme activity, mitochondrial function, and enhanced engraftment in a liver injury mouse model. To evaluate translational relevance, mature hepatocytes were incorporated into extrusion-based 3D bioprinted constructs, which maintained hepatic viability and function. This integrated strategy of combining progenitor cell banking, zonation control, and tissue engineering offers a scalable and clinically relevant approach for generating functional human liver tissue suitable for therapeutic development.

cell biology↗

bioRxiv2025

Automatically Defining Protein Words for Diverse Functional Predictions Based on Attention Analysis of a Protein Language Model

Understanding the relationship between protein sequence and function remains a longstanding challenge in bioinformatics, and to date the lions share of related tools parse proteins at the domain or motif levels. Here, we define "protein words" as an alternative to "motif" for studying proteins and functional prediction applications. We first developed an unsupervised tool we term Protein Wordwise, which parses analyte protein sequences into protein words by analyzing attention matrices from a protein language model (PLM) through a community detection algorithm. We then developed a supervised sequence-function prediction model called Word2Function, for mapping protein words to GO terms through feature importance analysis. We compared the prediction performance of our protein word-based toolkit with a motif-based method (PROSITE) for multiple protein function datasets. We also assembled a functionally diverse data resource we term PWNet to support evaluation of protein words for predicting functional residues across 10 tasks (e.g., diverse biomolecular binding, catalysis, and ion-channel activity). Our toolkit outperforms PROSITE in all the examined datasets and tasks. By abandoning domains and instead using attention matrices from a PLM for automatic, systematic, and annotation-agnostic parsing of proteins, our toolkit both outperforms currently available tools for functional annotations at the residue and whole-protein levels and suggests innovative forms of protein analysis well-suited to the post-AlphaFold era of biochemistry.

bioinformatics↗

bioRxiv2024

A regulator controls both DNA damage response and anti-phage defense networks in Moraxellaceae

DNA damage chemicals, including many antibiotics, often induce prophage induction and phage outbreaks in microbial communities, a major threat to bacterial survival. Moraxellaceae contains clinically highly relevant strains with outstanding antibiotic and radio resistance, yet their cellular-level regulation in DNA damage response and anti-phage defense is largely unknown. Here, we identified a WYL family protein that had replaced the ubiquitous SOS system in evolution and directly regulated a transcriptional network of both DNA repair genes and anti-phage defense genes under DNA damage stress. This study depicts a mechanism that how bacteria maintain immunity to phages without compromising on antibiotic resistance, and sheds light on controlling the resistance of Moraxellaceae strains in clinical practice.

microbiology↗

bioRxiv2024

Organic opto-nanobiointerface enables multiscale biomodulation

Virtually all organic material on Earth has been produced converting solar energy through photosynthesis in chloroplasts, a sack-like, double membrane organelle in plants and algae, where transmembrane electron transfer occurs from lumen to stroma. Although animals hardly harness the power of photosynthesis, their bioelectrical signals extensively regulate complex electrophysiological behaviors, rendering it a superior target for biomedical innovation. Here, a crude structural mimicry of chloroplast has led us to discover that hollow sphere graphitic carbon nitride nanoparticles (hg-C3N4 NPs) endowed non-genetic, subcellular and intercellular photo-modulation of various excitable and non-excitable cells, accumulatively achieving modulation at tissue/organ function level. The homogeneous hg-C3N4 NPs showed responsiveness to light via both photoelectrochemical and photothermal mechanisms. The hg-C3N4 NPs can be spontaneously internalized with excellent cytocompatibility. Using a focusing laser, the hg-C3N4 NPs enable intracellular optical stimulation with subcellular resolution, inducing calcium transient release in multiple cells and propagation in primary cardiomyocytes and cardiac fibroblasts. At multicellular scale, optical pacing and synchronization of cardiomyocyte beating is readily achieved by LED. Further, we demonstrate that hg-C3N4 nanoparticles can be safely delivered into the mouse eye and elicit measurable cortical and behavioral light responses in a subset of animals in a model of advanced retinal degeneration. Finally, application of hg-C3N4 NPs to porcine retinal tissue ex vivo confirmed their modulation capability to directly activate RGCs activity under LED photostimulation. Taken together, these nanostructured biomimetic photocatalytic NPs offer high resolution, leadless optical probing, non-invasive delivery and great biocompatibility, serving as a versatile tool for addressing a range of complex biomedical challenges through subcellular, intercellular and tissue-level photo-modulation across a broad spectrum of scales.

bioengineering↗

bioRxiv2024

Multi-View Integrative Approach For Imputing Short-Chain Fatty Acids and Identifying Key factors predicting Blood SCFA

Short-chain fatty acids (SCFAs) are the main metabolites produced by bacterial fermentation of dietary fiber within gastrointestinal tract. SCFAs produced by gut microbiotas (GMs) are absorbed by host, reach bloodstream, and are distributed to different organs, thus influencing host physiology. However, due to the limited budget or the poor sensitivity of instruments, most studies on GMs have incomplete blood SCFA data, limiting our understanding of the metabolic processes within the host. To address this gap, we developed an innovative multi-task multi-view integrative approach (M2AE, Multi-task Multi-View Attentive Encoders), to impute blood SCFA levels using gut metagenomic sequencing (MGS) data, while taking into account the intricate interplay among the gut microbiome, dietary features, and host characteristics, as well as the nuanced nature of SCFA dynamics within the body. Here, each view represents a distinct type of data input (i.e., gut microbiome compositions, dietary features, or host characteristics). Our method jointly explores both view-specific representations and cross-view correlations for effective predictions of SCFAs. We applied M2AE to two in-house datasets, which both include MGS and blood SCFAs profiles, host characteristics, and dietary features from 964 subjects and 171 subjects, respectively. Results from both of two datasets demonstrated that M2AE outperforms traditional regression-based and neural-network based approaches in imputing blood SCFAs. Furthermore, a series of gut bacterial species (e.g., Bacteroides thetaiotaomicron and Clostridium asparagiforme), host characteristics (e.g., race, gender), as well as dietary features (e.g., intake of fruits, pickles) were shown to contribute greatly to imputation of blood SCFAs. These findings demonstrated that GMs, dietary features and host characteristics might contribute to the complex biological processes involved in blood SCFA productions. These might pave the way for a deeper and more nuanced comprehension of how these factors impact human health.

bioinformatics↗

bioRxiv2024

Regulation of alternative polyadenylation isoforms of Timp2 is an effector event of RAS signaling in cell transformation

Alternative polyadenylation (APA) generates mRNA isoforms with different lengths of the 3 untranslated region (3 UTR). The tissue inhibitor of metalloproteinase 2 (TIMP2) plays a key role in extracellular matrix remodeling under various developmental and disease conditions. Both human and mouse genes encoding TIMP2 contain two highly conserved 3UTR APA sites, leading to mRNA isoforms that differ substantially in 3UTR size. APA of Timp2 is one of the most significantly regulated events in multiple cell differentiation lineages. Here we show that Timp2 APA is highly regulated in transformation of NIH3T3 cells by the oncogene HRASG12V. Perturbations of isoform expression with long 3UTR isoform-specific knockdown or genomic removal of the alternative UTR (aUTR) region indicate that the long 3UTR isoform contributes to the secreted Timp2 protein much more than the short 3UTR isoform. The short and long 3UTR isoforms differ in subcellular localization to endoplasmic reticulum (ER). Strikingly, Timp2 aUTR enhances secreted protein expression but no effect on intracellular proteins in reporter assays. Furthermore, downregulation of Timp2 long isoform mitigates gene expression changes elicited by HRASG12V. Together, our data indicate that regulation of Timp2 protein expression through APA isoform changes is an integral part of RAS-mediated cell transformation and 3UTR isoforms of Timp2 can have distinct impacts on expression of secreted vs. intracellular proteins.

molecular biology↗

bioRxiv2024

De novo Design of A Fusion Protein Tool for GPCR Research

G protein-coupled receptors (GPCRs) play pivotal roles in cellular signaling and represent prominent drug targets. Structural elucidation of GPCRs is crucial for drug discovery efforts. However, structural studies of GPCRs remain challenging, particularly for inactive state structures, which often require extensive protein engineering. Here, we present a de novo design strategy termed "click fusion" for generating fusion proteins to facilitate GPCR structural studies. Our method involves the rational design of structurally stable protein domains rigidly linked to GPCRs. The resulting fusion protein enhances the thermostability of the target GPCR and aids in determining GPCR structures via cryo-electron microscopy (cryo-EM). We further demonstrate that the designed fusion protein can be transferred among structurally similar GPCRs with minor adjustments to the linker region. Our study introduces a promising approach for facilitating GPCR structural studies and advancing drug discovery efforts.

biophysics↗

bioRxiv2024

The alternative polyadenylation regulator CFIm25 promotes macrophage differentiation and activates the NF-κβ pathway

Macrophages are required for our bodys development and tissue repair and protect against microbial attacks. We previously reported a crucial role for regulation of mRNA 3-end cleavage and polyadenylation (C/P) in monocyte to macrophage differentiation. The CFIm25 subunit of the C/P complex showed a striking increase upon differentiation of monocytes with Phorbol Myristate Acetate, suggesting that it promotes this process. To test this hypothesis, CFIm25 was overexpressed in two different monocytic cell lines, followed by differentiation. Both cell lines showed a significant increase in macrophage characteristics and an earlier slowing of the cell cycle. In contrast, depletion of CFIm25 hindered differentiation. Cell cycle slowing upon CFIm25 overexpression was consistent with a greater decrease in the proliferation markers PCNA and cyclin D1, coupled with increased 3UTR lengthening of cyclin D1 mRNA. Since choice of other poly(A) sites could be affected by manipulating CFIm25, we identified additional genes with altered use of poly(A) sites during differentiation and examined how this changed upon CFIm25 overexpression. The mRNAs of positive regulators of NF-{kappa}B signaling, TAB2 and TBL1XR1, and NFKB1, which encodes the NF-{kappa}B p50 precursor, underwent 3UTR shortening that was associated with increased protein expression compared to the control. Cells overexpressing CFIm25 also showed elevated levels of phosphorylated NF-{kappa}B-p65 and the NF-{kappa}B targets p21, Bcl-XL, ICAM1 and TNF- at an earlier time and greater resistance to NF-{kappa}B chemical inhibition. In conclusion, our study supports a model in which CFIm25 accelerates the monocyte to macrophage transition by promoting alternative polyadenylation events which lead to activation of the NF-{kappa}B pathway.

molecular biology↗

bioRxiv2024

NOK promotes tumorigenesis through coordinating epidermal growth factor receptor to boost the downstream signaling in breast cancer

BACKGROUNDEpidermal growth factor receptor (EGFR) forms a homodimer or heterodimer with other ErbB receptor family members to activate different downstream cytoplasmic signaling proteins during tumorigenesis. METHODSAdenovirus and lentivirus were used to overexpress or deplete NOK and/or EGFR to evaluate the phosphorylation of EGFR, the interaction of NOK-EGFR and their role in cell proliferation and metastasis. RESULTSEGFR heterodimerizes with NOK (also known as STYK1), a novel tyrosine kinase with a transmembrane domain, to promote tumorigenesis and metastasis of breast cancer cells. We found that NOK directly interacted with EGFR and formed a heterodimer complex. Depletion of NOK impaired, but over-expression of NOK increased, the phosphorylation of EGFR. NOK enhanced EGF signaling activation, in particular, the phosphorylation of STAT3, STAT5 and Erk1/2 via its juxtamembrane (JM) domain in promoting the proliferation and migration of breast cancer cells. Overexpression of NOK and EGFR synergistically induced the tumorigenesis of NIH-3T3 normal cells. We finally demonstrated that co-expression of NOK and EGFR correlated with tumor malignant stages in breast cancer patients. CONCLUSIONSOur findings uncover a mechanism by which NOK coordinates EGFR to enhance EGF signaling during tumorigenesis and metastasis and propose a potential strategy for targeting NOK-EGFR in breast cancer treatment. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/608018v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@17b0078org.highwire.dtl.DTLVardef@5bfe6forg.highwire.dtl.DTLVardef@19d3d15org.highwire.dtl.DTLVardef@155fc4_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIEGFR heterodimerizes with NOK/STYK1, a novel tyrosine kinase with a transmembrane domain, in a manner of cross interaction via their juxtamembrane (JM) domains and kinase domains. C_LIO_LINOK enhances EGF signaling activation, in particular, the phosphorylation of STAT3, STAT5 and Erk1/2 via its JM domain. C_LIO_LINOK and EGFR synergistically promote proliferation and migration of breast cancer cells and induce tumorigenesis of normal cells. C_LIO_LICo-expression of NOK and EGFR correlates with tumor malignant stages in breast cancer patients. C_LI

cell biology↗