Cytoskeleton reorganization induced by a novel K6-K14 keratin fusion promotes cancer stemness and cellular plasticity via cGAS-STING selection

Cytoskeleton reorganization induced by a novel K6-K14 keratin fusion promotes cancer stemness and cellular plasticity via cGAS-STING selection | 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 Cytoskeleton reorganization induced by a novel K6-K14 keratin fusion promotes cancer stemness and cellular plasticity via cGAS-STING selection Jim Sheu, I-Hsuan Chen, Senthilkumar Ravichandran, Ming-Tsung Lai, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6985645/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jan, 2026 Read the published version in Oncogenesis → Version 1 posted 10 You are reading this latest preprint version Abstract Cytoskeletal network dynamics play important roles in regulating cellular functions. Although alterations in cytoskeleton-related genes are frequently detected, limited attention has been paid to their roles in cancer development. A novel keratin fusion variant, K6-K14/V5, was previously identified in head and neck squamous cell carcinoma, and its expression led to catastrophic nuclear collapse, resulting in DNA breaks and cGAS-STING activation. Such cell-killing effects can trigger autophagy induction, which, in turn, promotes cancer cell evolution/clonal selection in a dormant state. Furthermore, due to the disrupted cellular architecture and the loss of mechanosensing, these dormant cells could only survive and adapt within a softer microenvironment, such as collagen gel. Upregulation of the partial epithelial-mesenchymal transition program by cytoskeleton reorganization was defined as a key step for these dormant cells to reactivate and regain their mechanical properties. Striking cell protrusions and increased MMPs were observed in the reactivated cells, facilitating interactions with the surrounding extracellular matrix and enhancing their invasive potential. Elevated extracellular vesicles were also found in the reactivated cells, which actively stimulated tumor growth via the FGF-FGFR axis. Our study offers a model for understanding how genetic alterations in cytoskeletal genes can directly contribute to cancer development and drive cancer evolution. Biological sciences/Cell biology/Cytoskeleton Biological sciences/Cancer/Oncogenes cytoskeleton cytokeratin keratin fusion cancer stemness cellular plasticity cGAS-STING dormancy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Significance Cytoskeletal reorganization induced by a novel keratin fusion variant, K6-K14/V5, not only alters the mechanical properties of cancer cells but also imposes selective pressure via cGAS-STING activation, promoting cancer aggressiveness. Introduction Cytoskeleton is a highly dynamic network that connects all organelles and components inside the cell. Assembly and disassembly of these filament proteins control many biological processes, including chromosome segregation/cell division, cell-cell/cell-matrix contact, cell migration/invasion, and transitions between epithelial and mesenchymal states. In particular, cells within tumor lesions are exposed to a high degree of heterogeneity with different mechanical properties [ 1 ]. During mechanotransduction, cytoskeletal proteins act as both receptors and targets to integrate mechanical signals with other cellular responses [ 1 – 4 ]. To adapt and survive, cancer cells actively respond to extracellular matrix (ECM) perturbations by transducing mechanical cues from adhesion molecules to the cell nucleus via the linker of the nucleoskeleton and cytoskeleton (LINC) complex, changing chromatin structure and gene expression [ 2 – 4 ]. Among the three major cytoskeletal networks, intermediate filaments (IFs) play a primary role in providing mechanical support to maintain cell shape and nuclear integrity. Changes in cellular architecture generate internal forces that cause mechanical stress and nuclear deformation. Disturbance of nuclear integrity also triggers genome instability and DNA breaks/damage, resulting in cGAS-STING activation and chronic inflammation [ 5 , 6 ]. Type I interferons (IFNs) are the main downstream effectors, contributing to cell senescence, autophagy, or cell death [ 5 , 6 ]. Thus, long-term cGAS-STING activation driven by genome instability is considered as a selection barrier to enrich cell clones with higher aggressiveness, a mechanism by which cancer cells escape immune surveillance and metastasize [ 5 – 7 ]. These findings suggest a molecular conversion of mechanical stress-induced DNA damage response (DDR) into aggressive cancer phenotypes through evolutionary adaptation. (Cyto)keratins are key components of IF networks with high tissue-/cell-type specificity [ 8 , 9 ]. Alterations in keratin profiles have been associated with epithelial-mesenchymal transition (EMT) and cancer stem cell formation, contributing to tumor progression, drug resistance, and metastasis [ 4 , 8 – 10 ]. Furthermore, destabilization of keratin networks or upregulation of their turnover can reduce cell-cell or cell-matrix contact, promoting cell migration [ 8 , 9 ]. In a previous study, we identified several novel keratin fusions in head and neck squamous cell carcinoma (HNSCC), and their presence correlated with cancer progression and poor clinical outcomes [ 11 ]. Notably, inter-keratin fusions can be detected in the adjacent normal-like epithelia, accounting for more than 30% of annotated in-frame fusions [ 11 ], suggesting their active roles in driving cancer development rather than merely as byproducts after cancer cell evolution. One K6-K14 fusion variant, K6-K14/V7, has been shown to promote cancer stemness through upregulating TGF-β and G-CSF signaling [ 11 ]. Additionally, its structure can interact with actin filaments and microtubules, significantly influencing the geometry of cytoskeletal networks [ 12 ]. In this study, we explored the functional consequences of nuclear deformation and DNA damages induced by another fusion variant, K6-K14/V5. Strikingly, this variant forms aggregates around the nucleus, altering cellular mechanical properties and disrupting the nuclear envelope. This study revealed a novel finding that K6-K14/V5-induced DDRs and the subsequent cGAS-STING activation can provide the advantages for cancer cells to acquire plasticity in cellular adaptation and clonal evolution, promoting aggressive cancer phenotypes through cytoskeleton reorganization. Materials and Methods Cell lines and reagents HNSCC cell lines Cal27 and FaDu were obtained from the Bioresource Collection and Research Center, Taiwan. Human oral fibroblasts (HOrFs) were purchased from ScienCell Research (Carlsbad, CA). All cells were maintained in DMEM (Gibco/Thermo Fisher Scientific, Waltham, MA) containing 10% FBS (Hyclone/Cytiva, Washington, DC) and 1% penicillin/streptomycin (Gibco/Thermo Fisher Scientific) at 37°C and 5% CO 2 in a humidified incubator. Antibodies used in this study are summarized in Table S1 . MMP Array GS1 was purchased from RayBiotech (Norcross, GA). FGFR inhibitors BGJ-398 (HY-13311) and AZD4547 (HY-13330) were purchased from MedChemExpress (Monmouth Junction, NJ). Gene constructs, stable cell clones and extracellular vesicles (EVs) Gene segments of K14 and K6-K14/V5 were amplified from cDNAs prepared from HNSCC samples as previously described [ 11 ]. SUN2 and TUBG genes were amplified from Cal27 cells and sub-cloned into the pmCherry-N1 vector (Clontech, Madison, WI). Stable cell clones were generated in Cal27 and FaDu cells by G418 selection after gene transfection. For cell clones stably expressing K6-K14/V5, transfected cells were grown on dishes covered with collagen-I (A1048301; Thermo Fisher Scientific). Cell images were analyzed using Image-Pro Premier (Media Cybernetics, Rockville, MD) or ImageJ (NIH). To analyze the EVs, 70% confluent cells were washed with PBS and cultured in serum-free Opti-MEM (Thermo Fisher Scientific) for 48-hr. The conditioned media were collected and centrifuged to remove debris, followed by concentration using Amicon ultra columns (Merck Millipore, Burlington, MA). The concentrates were applied onto qEV size exclusion columns (Izon Science, Christchurch, New Zealand) for EV separation. Size distributions and EV concentrations were analyzed using an Exoid TRPS nanoparticle analyzer (Izon Science). Assessments of mechanical properties and cell topology One day before the measurement, cells were seeded into 10 cm dishes at a density of 3–5×10 3 cells/cm 2 for 24-hr in phenol red-free DMEM. A JPK NanoWizard-II Atomic Force Microscope (AFM) with BioCell (JPK Instruments, Berlin, Germany) was equipped to detect the nuclear stiffness of cell clones as previously described [ 13 ]. Cell topology study was carried out by scanning over areas of 100×100 µm in contact mode using an FV1000 confocal microscope (Olympus, Shinjuku, Japan). The average cell height was calculated from 15 cells per stable clone and presented as mean ± standard deviation (SD). Determination of DNA content loss Cells were stained with cell-permeable DAPI (#268298; Sigma-Aldrich, Darmstadt, Germany) and investigated under an FV1000 confocal microscope. The nuclear volume and DNA content were calculated using DAPI images generated by sequential z-series scans. Cavitation of the cell nucleus was visualized based on the DAPI intensity. Alternatively, cells were stained with a mixture of RNase (10 µg/mL) and propidium iodide (50 µg/mL), and the DNA content (at 617 nm) was analyzed using an LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ). Additionally, a comet assay was performed to detect DNA breaks (ab238544; Abcam). Immunofluorescence staining and imaging Cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. After blocking (10% FBS, 0.3% Triton X-100, 100mM Tris, 150mM NaCl, pH = 7.5) for 1-hr, the cells were incubated with primary antibodies (Table S1 ) at 4°C overnight. On the second day, the cells were washed and stained with secondary antibodies for 1-hr. For actin staining, cells were washed with warm PBS, fixed, and stained with Rhodamine-conjugated phalloidin (R415; Thermo Fisher Scientific). NIR-Cytopainter (ab176833; Abcam, Cambridge, UK), was used for mitochondrial staining. The cells were counterstained with DAPI and visualized under an IX83 fluorescence microscope (Olympus). Actin alignment score (AAS) The angular distribution of stress fibers was analyzed using AAS, a parameter modified from a previous study [ 14 ]. In brief, the images of each stress fiber (length > 10µm) were individually extracted. Filament angles to migration direction were estimated using CDC42 as the polarization marker, combined with the largest cell length. Then, every angle was converted into its corresponding Tensor, where Tensor = RADIANS(filament angle – the average angle in one cell). AAS = 100×Cos(radius). The average AAS was calculated from 15 cells per stable clone and presented as mean ± SD. Cell-based functional studies Cell proliferation was assessed by MTT assay. For 3D co-culture study, 10 4 mCherry-expressing CAL27 cells were mixed with equal amounts of cells expressing different constructs and seeded into a hanging drop plate (3D Biomatrix, Ann Arbor, MI). Cell images were taken after 10-days and the sphere sizes were analyzed using Image-Pro Premier. To assess cell stemness, limiting dilution analysis was performed by seeding cells in 96-well ultra-low attachment plates (Corning, Avon, France) at densities of 1, 10, 100, or 1000 cells/well. After 10-days, visible spheres (diameter > 20µm) were counted in each well. The sphere formation activity was calculated using ELDA (bioinf.wehi.edu.au/software/elda/) and quantified as scores for sphere-forming efficiency (SFE) and cancer-initiating cells (CIC). Cell invasiveness was assessed using Boyden chamber (Merck Millipore). Additionally, THUNDER live cell system (Leica Microsystems, Wetzlar, Germany) was utilized to decode the invadopodia of cells grown on collagen, labeled with Alexa-Fluo-568 NHS Ester (A20103; Thermo Fisher Scientific). To study stroma-dependent cell growth, 3×10 5 HOrFs were seeded on NaOH-pretreated coverslips in 24-well plates. After 48-hr, 10 5 Cal27 cells per well were seeded on top of the HOrFs and cultured for 3-days in the presence or absence of 1 µM FGFR inhibitors. Cells on the coverslips were fixed and stained using a Masson Tricolor Staining Kit (G1006; Servicebio, Wuhan, China). Transcriptome analysis RNA samples were extracted from cells transiently or constitutively expressing gene constructs using an RNeasy Kit (Qiagen, Hilden, Germany). To study intercellular communication, RNA samples were also prepared from HOrFs treated with conditioned media for 48-hr. Samples from transiently transfected cells were sent for microarray analysis (Clariom_D_Human) at the Genomics Center, Kaohsiung Chang-Gung Memorial Hospital, Taiwan. RNA samples from the stable cell clones or educated HOrFs were sent for RNA sequencing (RNA-seq) service at Biotools (Taipei, Taiwan), utilizing NovaSeq 6000 platform (Illumina, San Diego, CA). Gene-set enrichment analysis (GSEA) was performed on the processed microarray or RNA-seq data using GSEA Java (version-4.3.3) from the Broad Institute. The functional interactome of upregulated and downregulated genes was analyzed using STRING protein-protein interaction database (string-db.org/). TOPCONS (topcons.cbr.su.se/) was utilized to define secreted or membrane proteins [ 15 ], and CellChatDB dataset (cellchat.org/cellchatdb/) was utilized to clarify the intercellular communications [ 16 ]. Ultrastructural analysis by transmission electron microscope (tEM) Unless specified, all chemicals in this study were purchased from Sigma-Aldrich. Cells were cultured in suspension (5×10 5 cells) in 10 cm Petri dishes. After 48-hr, the spheroids were collected by centrifugation, followed by fixation with 2.5% glutaraldehyde (G5882) in 0.1M cacodylate buffer (C4946; pH 7.4) for overnight at 4°C. After washes, the spheroids were immersed with 1% OsO 4 (#201030) for 2-hr at 4°C and stained with UA zero EM (R1000; Agar Scientific, Essex, UK) for 1-hr. After washes with nanopure water, cells were dehydrated in graded ethanol solutions from 50–100%. An epoxy-embedding kit (#45359) was used to embed the samples in beem capsules, then cured at 45°C for 24-hr and 60°C for 48-hr. Sections (50–70 nm) were prepared and placed on 100 mesh copper grids for image observation using an HT7500 tEM (Hitachi High-Tech, Tokyo, Japan). Tumor xenografts in nude mice Six-week-old male athymic nu/nu mice were purchased from the National Laboratory Animal Center, Taiwan and randomly assigned into groups. Stable cell clones were mixed with matrigel (1:1 v/v) (DLW356231; Sigma-Aldrich) and injected subcutaneously into mice (5×10 6 cells per injection, two sites per mouse, 5 mice per group). Sixty-days after cell inoculation or when the tumor size reached 3000 mm³, the mice were euthanized. Tissue sections were prepared for histological examination using previously described protocols [ 11 ]. All studies were conducted under the guidelines of the Institutional Animal Care and Use Committee at National Sun Yatsen University (IACUC-10909 and − 11126). Statistical analyses Statistical analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA) and SPSS (version-14.0; SPSS Inc., Chicago, IL). A t -test or Fisher’s exact test was used for comparisons between two groups, whereas one-way ANOVA was used for multiple group comparisons. A p -value significance was set at p < 0.05. Results Acute K6-K14/V5 expression triggers nuclear deformation and DNA breaks, leading to cGAS-STING activation We have previously identified seven major K6-K14 keratin fusions in HNSCC [ 11 ]. Among them, the K6-K14/V5 variant tends to form aggregates prominently around the cell nucleus, resulting in the complete loss of wild-type keratin network within cells. To investigate its functional roles in cancer development, gene constructs were expressed in Cal27 and FaDu cells which show undetectable fusion events (Figure S1 A). Dislike clear IF network formed by K14, K6-K14/V5 expression led to aggregates, accompanied by higher frequencies of nuclear deformation and micronuclei in both cell lines (Fig. 1 A, Figure S1 B). By co-transfecting mCherry-tagged SUN2, a key component of the LINC complex, K6-K14/V5 expression showed significant damage to the nuclei (Figure S2 A). Cell topography revealed that K6-K14/V5 exerted unbalanced mechanical forces, leading to nuclear distortion and the formation of hollow nuclei (Figure S2 B, Figure S2 C). The DNA content was reduced accordingly (Figure S2 B) with a notable reduction in nuclear volume (Figure S2 D) and stiffness (Figure S2 E). Since DNA damage can cause centrosome disorganization [ 17 ], K6-K14/V5 was found to form aggregates with centrosomes with increased total number (Figure S2 F). Comet assay clearly detected DNA breaks in cells expressing K6-K14/V5 (Fig. 1 B), associated with the formation of phospho-CHK2 (p-CHK2) and γH2AX foci in the nuclei in a time-dependent manner (Fig. 1 B). These findings suggest the activation of ATM/ATR-mediated DDRs via the p53-p21 cascade, as verified by western blotting (Fig. 1 C). Increased levels of γH2AX ubiquitination, a marker for active induction of DNA lesions [ 18 ], were also observed. Cell proliferation was significantly suppressed which can be partially rescued by an ATM/ATR inhibitor CGK733 (Fig. 1 D). Among the 12 upregulated reactomes (genes with fold change > 2.0 compared to K14 control), three indicated the involvement of cytosolic DNA-induced IFN signaling (Figure S3). Immunostaining further confirmed the activation of cGAS-STING signaling associated with the DDRs (Fig. 1 E). Acute K6-K14/V5 expression reduces mechanosensing and alters the preference for matrix rigidity The LINC complex is a central component of mechanosensing, thus K6-K14/V5 expression might influence the mechanical properties. F-actin staining revealed well-organized networks in the control cells, enabling cell extension and spreading. In contrast, less cell protrusion with reduced focal adhesion formation and spreading area was observed in cells acutely expressing K6-K14/V5 (Fig. 2 A), suggesting less intracellular tension and force transmission [ 19 ]. With reduced cortical actomyosin contractility, topological study by atomic force microscope (AFM) confirmed increased cell height in cells expressing K6-K14/V5 (Fig. 2 B). Confocal microscopy further revealed relaxed cell nuclei with expansion in the z-direction (Fig. 2 B). Western blotting indicated the downregulation of mechanosensing, including focal adhesion-mediated integrin signaling (p-ITGB1, p-FAK, p-PI3K, and p-MAPK1) and intracellular contractility (PAK1 and myosin IIb) (Fig. 2 C). These findings are very similar to those reported in a previous study that disruption of keratin stability by overexpressing a dominant K14 mutation R416P inhibited the normal mechanical response to substrate rigidity, reducing F-actin stress fibers and cell stiffness [ 20 ]. Because changes in rigidity sensing and matrix preference is a mechanical phenotype during cell transformation [ 13 , 21 ], we compared the cell growth ability by seeding K6-K14/V5-expressing cells on regular culture dishes or dishes covered with collagen gels under G418 selection. As expected, K6-K14/V5-positive cells lost their population after several passages on regular dishes, whereas the same transfected cells could survive on the collagen and finally became the major population, named as 3D-V5 cells (Fig. 2 D). Strikingly, the surviving 3D-V5 cells also changed their morphology after two weeks and formed sphere-like aggregates (Fig. 2 D). Induction of cancer dormancy via autophagy in the surviving 3D-V5 cells Notably, the surviving 3D-V5 cells constitutively activated IFN-mediated signaling (Figure S4) and did not abate cGAS-STING signaling as previously proposed [5. 6], suggesting the existence of other mechanisms to subvert lethal epithelial responses to cytosolic DNA [ 6 , 7 , 22 ]. For this, gene-set enrichment analysis (GSEA) revealed some enriched pathways contributing to cancer dormancy, including downregulation of cell cycle progression, focal adhesion-mediated signaling, ERK/MAPK signaling, and immunogenic potentials (Figure S4). In line with these, upregulation of drug resistance and retinoic acid signaling were also observed. Several studies have shown autophagy as a key mechanism for cancer cells to overcome cellular stresses via dormancy induction [ 22 , 23 ]. In our study, several genes involved in autophagic process were found upregulated in 3D-V5 cells (Fig. 3 A; Table S2 ), which can be confirmed by immunostaining and western blotting (Fig. 3 B and 3 C). Ultrastructural analysis also revealed an increase in mitochondrial fission, resulting in elevated small mitochondria with some in autophagic vacuoles (Fig. 3 D). Upregulation of metabolic reprogramming processes associated with autophagy-induced cell dormancy was also found in 3D-V5 cells (Figure S4). Among them, pathways contributing to vitamin A-retinoic acid signaling were most significantly activated (Fig. 3 E), which regulates stem cell dormancy and cellular plasticity via remodeling spatial chromatin context [ 24 , 25 ]. Long-term K6-K14/V5 expression reprograms keratin expression profiles with a partial-EMT (pEMT) phenotype Considering DDR-induced catastrophe with a self-defined fitness may serve as selective pressure for cancer evolution [ 5 – 7 , 26 ], we then determined whether the surviving 3D-V5 cells show different phenotypes from those with acute K6-K14/V5 expression. First, 3D-V5 cells could survive on a regular culture dish and partially recover the IF network with visible nuclear array. The LINC complex maintained in a compact shape, although some were empty without detectable DNA (Fig. 4 A). GSEA revealed keratinization as the most upregulated pathway in 3D-V5 cells as compared to controls (Fig. 4 B, upper ; Figure S4), indicating the assistance of additional keratin members to reorganize K6-K14/V5 network in the surviving cells. Several altered keratins have been linked to an aggressive program for epithelial tumors, including upregulation of K19, K14, K15, K6, and K17, as well as downregulation of K8/K18 (Fig. 4 B, lower ) [ 4 ]. To determine the stemness potential, we screened the expression levels of gene signatures for epithelial (E) and mesenchymal (M) phenotypes in HNSCC [ 27 , 28 ]. 3D-V5 cells tended to gain a higher E-score than control cells (Fig. 4 C), however, they did not show increased levels of CDH1 and EPCAM. In contrast, certain mesenchymal genes were upregulated (e.g., EMILIN1, LAMA4, FOXC2, MMP2, and ITGA5), whereas an overall lower M-score was found. Those data suggest a transition state between full-E and full-M programs. To confirm this, we analyzed gene signatures for pEMT program in HNSCC [ 28 ]. K14-expressing cells showed a higher pEMT score than GFP cells as previously proposed [ 28 – 30 ]. Nevertheless, 3D-V5 cells harbored an even higher pEMT potential with extremely high levels (more than 2 6 folds) of MMP10, ACKR3 (CXCR7), and SOX2 (Fig. 4 C). When co-cultured with mCherry-expressing parental cells in hanging drops, 3D-V5 cells localized to the surface of cell spheres and generated a unique microenvironment to nurture the sphere growth (Fig. 4 D), a potent feature for HNSCC cells at pEMT state [ 28 ]. Although K14-expressing cells promoted sphere growth, they did not form a clear “cell shell” outside the sphere. Through limiting dilution assay, increased stemness and plasticity were confirmed in 3D-V5 cells (SFE: 24.5% and CIC: 1/8.2), as compared to K14- (SFE: 12.3% and CIC: 1/51.0) and GFP-expressing (SFE: 6.5% and CIC: 1/80.9) cells (Fig. 4 E). These data suggest that long-term expression and selection by K6-K14/V5 promotes cancer aggressiveness by upregulating pEMT program through keratin network reorganization. Surviving 3D-V5 cells regain mechanical strength to promote cell protrusion and invasion In addition to oncogenic keratins, several genes related to actin and microtubule networks were also upregulated, which form compact interactomes for cytoskeleton organization and actin-mediated cell contraction, suggesting the recovery of mechanical properties in the surviving cells (Fig. 5 A; Table S3). Consistently, the nuclear stiffness of 3D-V5 cells was found to increase (Fig. 5 B). As previously shown, overexpression of Piezo2 mechanosensitive channel is essential to enhance mechano/biological responses involved in cell-matrix sensing and cellular adaptation in the tumor microenvironment (TME) [ 31 ], which could also be detected in 3D-V5 cells (Fig. 5 C). The downstream effectors of Pieozo2-mediated Ca 2+ -influx were also upregulated (Fig. 5 C). Actin filament staining revealed increased stress fibers (Fig. 5 D) and a higher alignment value (Fig. 5 E) in 3D-V5 cells, suggesting the strengthening ability to migrate and invade [ 14 ]. When grown in a 3D microenvironment, 3D-V5 cells, especially those on the surface layer of the spheres, showed striking cell protrusions, supporting the pEMT phenotype for active interactions with the surrounding ECM (Fig. 5 F). Additionally, data from both transcriptome and protein array studies confirmed upregulation of MMP1, MMP10, and MMP13 in 3D-V5 cells, which associated with the downregulation of their inhibitor, TIMP-2 (Fig. 5 G and 5 H). When cultured on collagen gel, enhanced invadipodia protrusion was found in 3D-V5 cells (Fig. 5 I). Transwell assay further indicated a significantly increased ECM-invasion ability (Fig. 5 J). These data suggest enhanced mechanosensing and invasiveness in 3D-V5 cells via cytoskeleton reorganization. Surviving 3D-V5 cells promote tumor formation via extracellular vesicle (EV)-mediated intercellular interactions When observed under tEM, we noticed elevated EVs secreted from 3D-V5 cells (Fig. 6 A). EV isolation and quantification further confirmed this finding (Fig. 6 A), suggesting active intercellular interactions. As compared to control cells, more genes were upregulated in 3D-V5 cells, especially the ones annotated as membrane-bound or secreted proteins (Fig. 6 B) [ 15 ]. When culturing human oral fibroblasts (HOrFs) with conditioned media, more expression alterations were found in 3D-V5-educated HOrFs (Fig. 6 B). Through ligand-receptor pairing [ 16 ], the major intercellular communications were mediated by those related to tumor growth, cancer-associated fibroblast (CAF) activation/proliferation, ECM remodeling, drug resistance, and neuron axon guidance (Fig. 6 C; Figure S5). Notably, PLXNA4 was overexpressed in treated HOrFs, and the expression of its receptor, SEMA6A, was enhanced in both 3D-V5 cells and treated HOrFs. Since SEMA6A also acts as a ligand for PLXNA4, the PLXNA4-SEMA6A interaction can promote cell activation/proliferation in both cancer cells and CAFs [ 32 , 33 ]. Overexpression of ERBB2, FGFR1, and FGFR3 were also found in 3D-V5 cells, which are known to be co-activated by SEMA6A [32. 33]. Moreover, 3D-V5-educated HOrFs showed increased production of FGF10, FGF17, KLRD1, and CXCL12, contributing to FGFR1/FGFR3 signaling activation (Fig. 6 C; Figure S5). FGF13 and FGF22 were also overexpressed in 3D-V5 cells, serving as autocrine/paracrine factors to enhance FGFR signaling. Consistently, 3D-V5 cells exhibited a significantly higher proliferation rate than control cells when co-cultured with HOrFs (Fig. 6 D), which could be partially suppressed by selective FGFR inhibitors, BGJ-398 and AZD4547 (Fig. 6 D). In a mouse xenograft model, GFP-expressing cells formed well-differentiated lesions with clear keratin pearls, and K14-expressing cells generated moderately differentiated lesions with viable cancer cells arranged in the nests (Fig. 6 E). Dramatically, 3D-V5 lesions showed a poorly-differentiated phenotype, exhibiting variations in cell size and shape along with an infiltrative pattern (Fig. 6 E). Especially, cancer cells in the center of the lesion show active cannibalistic activity, which is frequently coupled with extracellular acidosis [ 34 , 35 ]. This finding suggests that degradation of the “loser” cells in acidic conditions could provide sufficient nutrients to nurture the “winner” cells with higher cancer stemness (CD44 + ) and proliferation rate (Ki-67 + ) (Fig. 6 E). Intense CD44 staining was also detected at the invasive front of 3D-V5 lesions, suggesting active interactions between “winner” cells and the ECM (Fig. 6 F). Approximately 40% (4 out of 10) of 3D-V5 lesions showed high CD44 expression and invasiveness to metastasize from subcutaneous layer into peritoneal cavity (Fig. 6 E and 6 G). Strong FGFR1/FGFR3 activation can be detected the interface between tumor lesions and the ECM (Fig. 6 H), guiding 3D-V5 metastasis. Discussion In this study, we investigated the molecular complexity of keratin IF networks in cancer cells and their impact on cellular adaptation and clonal selection/evolution following a nuclear catastrophe induced by K6-K14/V5 expression. By promoting autophagy, K6-K14/V5-expressing cancer cells with high genome instability can survive on collagen gel in a pro-dormant state, while altering their transcriptional profiles. As a result, the surviving cancer cells exhibit enhanced epithelial-mesenchymal dynamics and acquire cancer stemness. Since IFs are key components of the cellular architecture, K6-K14/V5-induced IF network disruption and the subsequent cytoskeleton reorganization provide cancer cells with elevated plasticity, allowing them to adapt to diverse ECM. A complex tumor ecosystem with active crosstalk between cancer and stromal cells can be established, ultimately leading to the reactivation of cancer dormancy and tumor spread. Our study presents a novel model for exploring the impact of changes in the mechanical properties of cells on cancer plasticity and complexity. Autophagy can trigger cancer dormancy to escape dead signaling, alter gene expression profiles, and promote metabolic reprogramming, a survival strategy for cancer cells to adapt and regrow by genetic or epigenetic alterations through a Darwinian evolutionary process [ 34 ]. Based on this perspective, genome instability triggered by K6-K14/V5 expression may function as an accelerator to generate cell clones with high heterogeneity, allowing them to evolve greater adaptability. Significantly, mechanical cues via the interactions with ECM play both positive and negative roles in regulating autophagic processes by controlling integrin-mediated and YAP/TAZ signals [ 35 ]. In our study, elevated EVs were released from the surviving 3D-V5 cells, leading to the accumulation of a variety of FGF ligands, collagens, and proteoglycans in the ECM. This may establish a positive feedback loop in which more alterations in the ECM can enhance FGF/FGFR signaling [ 36 ], resulting in more active cellular responses in ECM remodeling. Whether such interactions between 3D-V5 cells and stromal cells can feedback control autophagic statuses of the cancer cells will be an interesting topic to explore. FGF/FGFR signaling plays an important role in regulating cellular plasticity and promoting tumor recurrence; thus, it has been suggested as a potent target for treating patients with metastatic cancers [ 36 , 37 ]. Recent studies also support its involvement in HNSCC development and the therapeutic potential for patient treatment [ 38 ]. Nevertheless, the FGF-FGFR axis is just one of the key signaling pathways involved in the interactions between 3D-V5 cells and the ECM. Other factors contributing to CAF activation and dormancy reactivation need to be further defined. For examples, several matrix proteins, such as DCN, COL14A1, and COL15A1, were highly expressed in 3D-V5-educated HOrFs. These proteins have been reported to play dual roles in both tumor suppression and cancer progression, influencing the onset of cancer metastasis via ECM remodeling [ 39 – 42 ]. Further studies are needed to explore how mechanical plasticity in cancer cells may contribute to ECM remodeling in controlling cancer dormancy reactivation. A pEMT program at the leading edge of a tumor lesion has been suggested as an independent predictor of nodal metastasis, tumor grade, and adverse pathologic features in many cancer types, including HNSCC [ 28 – 30 ]. With increased mechanical complexity, cancer cells can easily stretch out and form invasive tips, allowing cells to break away through gaps within tissues and start to metastasize [ 43 ]. In our study, invasive tips can be frequently detected in these cells both in vitro and in vivo with higher migration and invasion ability in the surviving 3D-V5 cells with Piezo2 upregulation. In addition, 3D-V5 cells upregulated MMP1 and MMP10, which play roles in the collective detachment of cancer cells from the primary tumor site during distant metastasis [ 44 ]. Notably, MMP10 is known to maintain a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in cancer lesions [ 45 ]. These findings support the previous studies that dormant disseminated tumor cells share several similar phenotypes as cancer stem/progenitor-like cells [ 46 , 47 ]. By promoting active cross-talk between cancer cells and the TME through FGFR signaling, dormant cancer cells can gain the ability to reactivate and metastasize. This study has some limitations. First, we did not clearly know how mechanosensing loss by acute k6-k14/V5 expression triggers cytoskeletal reorganization and transcriptome reprogramming in the surviving 3D-V5 cells. Transposase-accessible chromatin with sequencing (ATAC-seq) may provide a raw map of chromatin connections to address the changes in the spatial chromatin context. Since k6-k14/V5 expression can alter the preference for matrix rigidity, it is also necessary to explore the mechanical impact of matrix rigidity on cellular adaptation and the signaling balance between cancer dormancy and cell awakening. Finally, S100A8, S100A9, and other S100 Ca 2+ -binding proteins were upregulated in the surviving 3D-V5 cells (Table S4), which have been known as potent attractants for neutrophil recruitment/activation [ 48 , 49 ]. Such immune editing can participate in different steps of tumorigenic processes, including cell motility/invasion/migration, cell cycle/cell proliferation, TME remodeling, drug resistance, and cancer stem cell formation [ 49 , 50 ]. Whether 3D-V5 directly influences neutrophil activity to facilitate cancer metastasis requires further investigation. Declarations Data availability Data from the experiments presented in this study are included in this published article and its Supplementary Information files. The transcriptome sequencing data are accessible at Gene Expression Omnibus (GEO) Databank (GSE292626). Competing interests: The authors declare no potential conflicts of interest. Acknowledgments All authors thank the technical assistance from Ms. Ting-Chia Chiang at National Sun Yatsen University/Taiwan. The authors also like to thank critical comments and suggestions from Prof. Tian-Li Wang at the John Hopkins Medical Institutions/USA, Prof. Dennis E. Discher at University of Pennsylvania/USA, and Prof. Chih-Wen Shu at National Sun Yatsen University/Taiwan. This study was supported by grants from National Science and Technology Council, Taiwan (112-2320-B-110-005-MY3, 109-2314-B-110-003-MY3, 113-2811-B-110-007), Kaohsiung Veterans General Hospital, Taiwan (KSVGH-113-101, KSVGH112-079), and Ministry of Health and Welfare, Taiwan (11126, 10916). Author contributions IHC: conceptualization, investigation, writing—original draft, writing—review & editing, and funding acquisition. 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Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupplTablesforK6K14V5.docx Supplmeentary Table S1 to S4 SupplFiguresforK6K14V5S1.docx Supplmeentary Fig.S1 to S5 Cite Share Download PDF Status: Published Journal Publication published 20 Jan, 2026 Read the published version in Oncogenesis → Version 1 posted Editorial decision: revise 05 Sep, 2025 Review # 1 received at journal 04 Sep, 2025 Review # 2 received at journal 27 Aug, 2025 Reviewer # 2 agreed at journal 16 Aug, 2025 Reviewer # 1 agreed at journal 16 Aug, 2025 Reviewers invited by journal 22 Jul, 2025 Submission checks completed at journal 11 Jul, 2025 First submitted to journal 10 Jul, 2025 Unknown event 27 Jun, 2025 Editor assigned by journal 26 Jun, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6985645","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":489195461,"identity":"9230c88d-abeb-486e-a983-6d077dc30bbe","order_by":0,"name":"Jim 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17:30:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6985645/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6985645/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41389-026-00598-1","type":"published","date":"2026-01-20T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87523298,"identity":"d82e86ac-c319-4817-bf34-805f608fc41c","added_by":"auto","created_at":"2025-07-24 18:29:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":817481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcute expression of keratin fusion variant K6-K14/V5 triggers nuclear deformation and DNA breaks. A\u003c/strong\u003eCal27 cells were transiently transfected with different expression constructs tagged with GFP for 24-hr. The K6-K14/V5 is a fusion variant detected in HNSCC with a junction site of K6A/18-K14/202 [11]. K14 was utilized as the backbone control. Nuclear deformation and micronucleus formation were detected by staining cells with DAPI and compared by bar charts. The degree of nuclear deformation was quantified by the vacant region within the nuclear circle area based on the DAPI image. \u003cstrong\u003eB\u003c/strong\u003e Cal27 cells transiently expressed different gene constructs were fixed at the indicated time points and stained with anti-phospho-CHK2 (p-CHK2) and anti-gH2AX antibodies. The representative images show the immunostaining results in cells 48-hr after the gene transfection (\u003cem\u003emiddle\u003c/em\u003e). In parallel, comet assay was performed 48-hr after the transfection (\u003cem\u003eleft\u003c/em\u003e). The positive cells with clear staining foci were counted at the indicated time points and averaged from 200 nuclei (\u003cem\u003eright\u003c/em\u003e). \u003cstrong\u003eC\u003c/strong\u003e Western blotting was conducted to analyze the possible activation of DDRs by using cell lysates from Cal27 cells transiently expressing K6-K14/V5. \u003cstrong\u003eD\u003c/strong\u003e Cell proliferation activity was estimated by MTT assay in cells expressing different constructs for 4 days (solid lines). ATM/ATR inhibitor CGK733 was added to K6-K14/V5-expressing cells to evaluate its rescue effect (dash line). \u003cstrong\u003eE\u003c/strong\u003e Immunostaining against cGAS and STING were performed to detect the activation of cGAS-STING signaling in cells transiently express different constructs for 48-hr. Data from each construct were compared to the ones from K14 control by \u003cem\u003et\u003c/em\u003e-test. For cell proliferation study in \u003cstrong\u003eD\u003c/strong\u003e, two-way ANOVA was applied to compare the statistical significance among different treatments. The \u003cem\u003ep\u003c/em\u003e-values were presented as *: \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05; **: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; and ***: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"FinalFiguresOncogenesiS11.png","url":"https://assets-eu.researchsquare.com/files/rs-6985645/v1/834437f20847bf459cb0f087.png"},{"id":87523300,"identity":"a5897847-1173-476a-b5fb-efd2eee9486d","added_by":"auto","created_at":"2025-07-24 18:29:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1014157,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eK6-K14/V5 expression alters the mechanosensing and matrix rigidity preference. A\u003c/strong\u003e Cal27 cells transiently expressed different gene constructs were fixed 48-hr after the gene transfection and stained with DAPI (blue), phalloidin labeling probe (red) or anti-vinculin antibodies (red). The spreading areas were estimated based on the area of phalloidin signal in each cell. \u003cstrong\u003eB\u003c/strong\u003eThe cell height and nuclear height in cells expressing different constructs were measured by AFM (\u003cem\u003eleft\u003c/em\u003e) and confocal microscope (\u003cem\u003eright\u003c/em\u003e), respectively. \u003cstrong\u003eC\u003c/strong\u003e Cal27 cells were transfected with K6-K14/V5 vector and the cell lysates were collected at the indicated time points for Western blotting. \u003cstrong\u003eD\u003c/strong\u003e Cal27 cells were transfected with K6-K14/V5-GFP vector and maintained in regular 2D culture dishes or dishes covered with collagen gels. The treated cells were enriched in the selection medium containing G418 (800 µg/ml) for one month. The cell images were taken under a fluorescent microscope every 4 days. Data from each construct were compared to the ones from K14 control by \u003cem\u003et\u003c/em\u003e-test in \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e. The \u003cem\u003ep\u003c/em\u003e-values were presented as *: \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05; **: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; and ***: \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"FinalFiguresOncogenesiS12.png","url":"https://assets-eu.researchsquare.com/files/rs-6985645/v1/763a55721c920bbb80e64f7f.png"},{"id":87523304,"identity":"fffc40fb-d26f-4f7a-a087-f996f522a053","added_by":"auto","created_at":"2025-07-24 18:29:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":705948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLong-term K6-K14/V5 expression promotes autophagic process and cancer dormancy.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e RNA samples were extracted from cell clones constitutively express GFP, K14-GFP and K6-K14/V5-GFP (the 3D-V5 cells), two clones for each construct, and subjected to transcriptome analysis by RNA-seq study. Genes involved in autophagic process and consistently upregulated (fold change \u0026gt; 1.2) or downregulated (fold change \u0026lt; 0.8) were colored in red and green, respectively, comparing to GFP and K14 controls (Table S2). \u003cstrong\u003eB\u003c/strong\u003e Imunostaining was performed to detect LC3 expression in cells stably express different gene constructs. \u003cstrong\u003eC\u003c/strong\u003e Western blotting was conducted to analyze the activation of key molecular events associated with autophagy. \u003cstrong\u003eD\u003c/strong\u003e Transmission electron microscope (tEM) was utilized to reveal the ultrastructural image of a 3D-V5 cell. The white arrow indicates the regular mitochondrion, and the yellow one indicates the small mitochondria resulting from mitochondrial fission. The magnified image from the yellow square shows two mitochondria undergoing mitophagy. \u003cstrong\u003eE\u003c/strong\u003e GSEA study reveals the enriched pathways contributing to vitamin A-retinoic acid signaling in 3D-V5 cells as compared to cells stably express GFP (\u003cem\u003eupper\u003c/em\u003e) or K14-GFP (\u003cem\u003elower\u003c/em\u003e). The databases used in this study include K: KEGG and R: Reactome. The statistical significances were presented as \u003cem\u003ep\u003c/em\u003e- or \u003cem\u003ep’\u003c/em\u003e-values by using GFP- or K14-expressing cells as the controls, respectively.\u003c/p\u003e","description":"","filename":"FinalFiguresOncogenesiS13.png","url":"https://assets-eu.researchsquare.com/files/rs-6985645/v1/47a7d4c97ec1a0079ab07b3a.png"},{"id":87523436,"identity":"6fa31214-5392-4d2c-b0cd-ede69f40c258","added_by":"auto","created_at":"2025-07-24 18:37:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":420773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D-V5 cells reorganize the keratin network and exhibit higher cancer stemness.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e The 3D-V5 cells were grown on a regular 2D culture dish and stained with anti-SUN2 antibodies for immunostaining study. The K6-K14/V5 network and LINC complex structure were visualized under a fluorescent microscope. The white triangle indicates a nuclear envelope-like structure without detectable DNA. \u003cstrong\u003eB\u003c/strong\u003e GSEA study reveals the enrichment of keratinization process based on Reactome database in 3D-V5 cells as compared to cells stably express GFP (\u003cem\u003eupper left\u003c/em\u003e) or K14-GFP (\u003cem\u003eupper right\u003c/em\u003e). The bar chart shows the fold changes of keratin genes involved in the keratin reorganization in 3D-V5 cells as compared to control cells expressing GFP or K14 (\u003cem\u003elower\u003c/em\u003e). \u003cstrong\u003eC\u003c/strong\u003e The heatmaps represent the expression levels of key markers for epithelial (E), mesenchymal (M), and partial-EMT (pEMT) programs in cells constitutively express different gene constructs, two cell clones for each construct, based on the RNA-seq data. The bar charts indicate the differences in E-score (the average log\u003csub\u003e2\u003c/sub\u003e(fold change of feature genes)), M-score, and pEMT-score among different groups. \u003cstrong\u003eD\u003c/strong\u003e Stable cell clones constitutively express different constructs were co-cultured with mCherry-expressing Cal27 cells in a 3D hanging drop condition for 10 days. The cell spheres were visualized under a fluorescent microscope, and sizes in diameter of entire spheres or parental spheres were measured by detecting the GFP and mCherry signals, respectively. \u003cstrong\u003eE\u003c/strong\u003e Cells expressing different constructs were seeded into 96-well U-bottomed ultra-low/non adherent plates at a cell density of 1, 10, 100, or 1000 cells per well and cultured for 10 days. The sphere-forming activity was observed under a fluorescent microscope and presented as the pie charts (\u003cem\u003eright\u003c/em\u003e). Cancer-initiating cell frequencies were computed and compared by Extreme Limiting Dilution Analysis (ELDA) software (\u003cem\u003eleft\u003c/em\u003e). The differences between 3D-V5 and control groups in \u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e were compared by\u003cem\u003e t\u003c/em\u003e-test. The statistical significances were presented as \u003cem\u003ep\u003c/em\u003e- or \u003cem\u003ep’\u003c/em\u003e-values by using GFP- or K14-expressing cells as the control, respectively.\u003c/p\u003e","description":"","filename":"FinalFiguresOncogenesiS14.png","url":"https://assets-eu.researchsquare.com/files/rs-6985645/v1/d690946f812cccbe07cb4805.png"},{"id":87523303,"identity":"0f5d0744-e8d2-4717-8cd1-be06d72b66a2","added_by":"auto","created_at":"2025-07-24 18:29:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":618967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D-V5 cells upregulate cytoskeleton reorganization and exhibit increased cell protrusion, migration and invasion. A\u003c/strong\u003e The interactome indicates the consistently upregulated genes in 3D-V5 cells as compared to GFP and K14 controls (fold change \u0026gt; 1.5), which participate in actin-binding, actin-mediated cell contraction, and microtubule organization (Table S3). \u003cstrong\u003eB\u003c/strong\u003e The nuclear stiffness of the stable 3D-V5 cell clones was detected by AFM. \u003cstrong\u003eC\u003c/strong\u003e The heatmap indicates the upregulation of genes involved in Ca\u003csup\u003e2+\u003c/sup\u003e-influx-induced actin remodeling in 3D-V5 cells as compared to GFP and K14 controls (fold change \u0026gt; 1.5). \u003cstrong\u003eD\u003c/strong\u003e Rhodamine-phalloidin was utilized to visualize the F-actin and stress fiber in cells stably express different gene constructs (\u003cem\u003eupper\u003c/em\u003e). Actin intensity and filament length were measured and averaged from 20 independent cells for each construct (\u003cem\u003elower\u003c/em\u003e). \u003cstrong\u003eE \u003c/strong\u003eActin alignment score (AAS) was estimated based on the alignments of actin fibers with the migration direction. The angular distribution of the stress fibers is displayed in a radar chart, where the values represent the frequencies of the stress fibers within 5-degree intervals, normalized by the total number of stress fibers. \u003cstrong\u003eF\u003c/strong\u003e Ultrastructural images of different cell clones grown as 3D spheres were captured using tEM. The expression profiles of MMP-related genes in cells expressing different constructs were analyzed by \u003cstrong\u003eG\u003c/strong\u003e RNA-seq or \u003cstrong\u003eH\u003c/strong\u003e protein arrays, and presented as fold changes using GFP clones as the control. \u003cstrong\u003eI\u003c/strong\u003e Cal27 cells expressing different constructs were grown on collagen gels containing fluorescent dye. Invadipodia protrusion of cells was analyzed by using THUNDER live-cell imaging system. \u003cstrong\u003eJ\u003c/strong\u003e The invasiveness of various cell clones was measured by using a Boyden chamber coated with matrigel (\u003cem\u003eleft\u003c/em\u003e). The average data were presented as means ± S.D. in a bar chart based on five independent experiments (\u003cem\u003eright\u003c/em\u003e). The GFP cell clones were utilized as the control in \u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e, \u003cstrong\u003eG\u003c/strong\u003e, \u003cstrong\u003eH\u003c/strong\u003e, and \u003cstrong\u003eJ\u003c/strong\u003e. The differences between two groups were compared by \u003cem\u003et\u003c/em\u003e-test. The statistical significances were presented as *: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; and ***: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"FinalFiguresOncogenesiS15.png","url":"https://assets-eu.researchsquare.com/files/rs-6985645/v1/ec74735c2894311ec3b944c5.png"},{"id":87523306,"identity":"e5195dba-af8c-45f1-a9bf-6c6a88952c6c","added_by":"auto","created_at":"2025-07-24 18:29:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3960359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D-V5 cells show enhanced interactions with ECM and exhibit higher cancer aggressiveness.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e The release of extracellular vesicles (EVs) from different cell clones was observed using tEM (\u003cem\u003eright\u003c/em\u003e). EVs were purified from the conditioned media of different cell clones. The Exoid TRPS system was utilized to quantify the concentration and size distribution of the collected EVs (\u003cem\u003eleft\u003c/em\u003e). \u003cstrong\u003eB\u003c/strong\u003e The bar chart on the \u003cem\u003eleft\u003c/em\u003e panel shows the numbers of significantly increased genes, log\u003csub\u003e2\u003c/sub\u003e(fold change) \u0026gt; 2, in the stable Cal27 cell clones across various comparisons. The bar chart on the \u003cem\u003eright\u003c/em\u003e shows the numbers of significantly altered genes in HOrFs treated with conditioned media from the stable Cal27 cell clones. \u003cstrong\u003eC\u003c/strong\u003e The Chord diagram displays the ligand-receptor interactions and their weights between the treated HOrFs (ligands) and 3D-V5 cells (receptors). \u003cstrong\u003eD\u003c/strong\u003e The HOrF-dependent cell growth was performed by culturing the stable Cal27 cell clones on confluent HOrFs for two days, followed by Masson's trichrome staining. \u003cstrong\u003eE\u003c/strong\u003e Tumor xenografts were generated in athymic \u003cem\u003enu/nu\u003c/em\u003e mice by subcutaneous injections of Cal27 cell clones expressing the indicated gene constructs. Mice were sacrificed on Day 60 and representative tumor sections were stained with H\u0026amp;E, anti-CD44, and anti-Ki67 Abs, respectively. The solid red dots indicate mice with distant metastasis. The magnified image from the red square shows the cells undergoing entosis (yellow arrows). Cancer tissues at \u003cstrong\u003eF\u003c/strong\u003e invasive front and \u003cstrong\u003eG\u003c/strong\u003e distant metastatic sites were stained with H\u0026amp;E and anti-CD44 Abs. \u003cstrong\u003eH\u003c/strong\u003e Cancer tissues at invasive front were stained with H\u0026amp;E, anti-phospho(p)-FGFR1, and anti-p-FGFR3 Abs.\u003c/p\u003e","description":"","filename":"FinalFiguresOncogenesiS16.png","url":"https://assets-eu.researchsquare.com/files/rs-6985645/v1/c3dbc521066455c3ed04d8e8.png"},{"id":101389655,"identity":"54f166c6-5402-44a6-82d7-9407fd78b7b2","added_by":"auto","created_at":"2026-01-29 08:06:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8693586,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6985645/v1/34cb1f13-8fe2-4c12-a37c-1c15d73ff012.pdf"},{"id":87523299,"identity":"7a77e079-48d7-454b-beee-44db1c2b1d9e","added_by":"auto","created_at":"2025-07-24 18:29:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32859,"visible":true,"origin":"","legend":"Supplmeentary Table S1 to S4","description":"","filename":"SupplTablesforK6K14V5.docx","url":"https://assets-eu.researchsquare.com/files/rs-6985645/v1/fba4260c2375863dced85f43.docx"},{"id":87523856,"identity":"ee34b4d6-2b23-4320-ad0c-91d315ef47cb","added_by":"auto","created_at":"2025-07-24 18:45:40","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12361391,"visible":true,"origin":"","legend":"Supplmeentary Fig.S1 to S5","description":"","filename":"SupplFiguresforK6K14V5S1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6985645/v1/4f679130f619014078e788af.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Cytoskeleton reorganization induced by a novel K6-K14 keratin fusion promotes cancer stemness and cellular plasticity via cGAS-STING selection","fulltext":[{"header":"Significance","content":"\u003cp\u003eCytoskeletal reorganization induced by a novel keratin fusion variant, K6-K14/V5, not only alters the mechanical properties of cancer cells but also imposes selective pressure via cGAS-STING activation, promoting cancer aggressiveness.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eCytoskeleton is a highly dynamic network that connects all organelles and components inside the cell. Assembly and disassembly of these filament proteins control many biological processes, including chromosome segregation/cell division, cell-cell/cell-matrix contact, cell migration/invasion, and transitions between epithelial and mesenchymal states. In particular, cells within tumor lesions are exposed to a high degree of heterogeneity with different mechanical properties [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. During mechanotransduction, cytoskeletal proteins act as both receptors and targets to integrate mechanical signals with other cellular responses [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. To adapt and survive, cancer cells actively respond to extracellular matrix (ECM) perturbations by transducing mechanical cues from adhesion molecules to the cell nucleus via the linker of the nucleoskeleton and cytoskeleton (LINC) complex, changing chromatin structure and gene expression [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmong the three major cytoskeletal networks, intermediate filaments (IFs) play a primary role in providing mechanical support to maintain cell shape and nuclear integrity. Changes in cellular architecture generate internal forces that cause mechanical stress and nuclear deformation. Disturbance of nuclear integrity also triggers genome instability and DNA breaks/damage, resulting in cGAS-STING activation and chronic inflammation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Type I interferons (IFNs) are the main downstream effectors, contributing to cell senescence, autophagy, or cell death [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, long-term cGAS-STING activation driven by genome instability is considered as a selection barrier to enrich cell clones with higher aggressiveness, a mechanism by which cancer cells escape immune surveillance and metastasize [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These findings suggest a molecular conversion of mechanical stress-induced DNA damage response (DDR) into aggressive cancer phenotypes through evolutionary adaptation.\u003c/p\u003e\u003cp\u003e(Cyto)keratins are key components of IF networks with high tissue-/cell-type specificity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Alterations in keratin profiles have been associated with epithelial-mesenchymal transition (EMT) and cancer stem cell formation, contributing to tumor progression, drug resistance, and metastasis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, destabilization of keratin networks or upregulation of their turnover can reduce cell-cell or cell-matrix contact, promoting cell migration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In a previous study, we identified several novel keratin fusions in head and neck squamous cell carcinoma (HNSCC), and their presence correlated with cancer progression and poor clinical outcomes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Notably, inter-keratin fusions can be detected in the adjacent normal-like epithelia, accounting for more than 30% of annotated in-frame fusions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], suggesting their active roles in driving cancer development rather than merely as byproducts after cancer cell evolution. One K6-K14 fusion variant, K6-K14/V7, has been shown to promote cancer stemness through upregulating TGF-β and G-CSF signaling [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, its structure can interact with actin filaments and microtubules, significantly influencing the geometry of cytoskeletal networks [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we explored the functional consequences of nuclear deformation and DNA damages induced by another fusion variant, K6-K14/V5. Strikingly, this variant forms aggregates around the nucleus, altering cellular mechanical properties and disrupting the nuclear envelope. This study revealed a novel finding that K6-K14/V5-induced DDRs and the subsequent cGAS-STING activation can provide the advantages for cancer cells to acquire plasticity in cellular adaptation and clonal evolution, promoting aggressive cancer phenotypes through cytoskeleton reorganization.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell lines and reagents\u003c/h2\u003e\u003cp\u003eHNSCC cell lines Cal27 and FaDu were obtained from the Bioresource Collection and Research Center, Taiwan. Human oral fibroblasts (HOrFs) were purchased from ScienCell Research (Carlsbad, CA). All cells were maintained in DMEM (Gibco/Thermo Fisher Scientific, Waltham, MA) containing 10% FBS (Hyclone/Cytiva, Washington, DC) and 1% penicillin/streptomycin (Gibco/Thermo Fisher Scientific) at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator. Antibodies used in this study are summarized in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. MMP Array GS1 was purchased from RayBiotech (Norcross, GA). FGFR inhibitors BGJ-398 (HY-13311) and AZD4547 (HY-13330) were purchased from MedChemExpress (Monmouth Junction, NJ).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGene constructs, stable cell clones and extracellular vesicles (EVs)\u003c/h3\u003e\n\u003cp\u003eGene segments of K14 and K6-K14/V5 were amplified from cDNAs prepared from HNSCC samples as previously described [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. SUN2 and TUBG genes were amplified from Cal27 cells and sub-cloned into the pmCherry-N1 vector (Clontech, Madison, WI). Stable cell clones were generated in Cal27 and FaDu cells by G418 selection after gene transfection. For cell clones stably expressing K6-K14/V5, transfected cells were grown on dishes covered with collagen-I (A1048301; Thermo Fisher Scientific). Cell images were analyzed using Image-Pro Premier (Media Cybernetics, Rockville, MD) or ImageJ (NIH). To analyze the EVs, 70% confluent cells were washed with PBS and cultured in serum-free Opti-MEM (Thermo Fisher Scientific) for 48-hr. The conditioned media were collected and centrifuged to remove debris, followed by concentration using Amicon ultra columns (Merck Millipore, Burlington, MA). The concentrates were applied onto qEV size exclusion columns (Izon Science, Christchurch, New Zealand) for EV separation. Size distributions and EV concentrations were analyzed using an Exoid TRPS nanoparticle analyzer (Izon Science).\u003c/p\u003e\n\u003ch3\u003eAssessments of mechanical properties and cell topology\u003c/h3\u003e\n\u003cp\u003eOne day before the measurement, cells were seeded into 10 cm dishes at a density of 3\u0026ndash;5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e for 24-hr in phenol red-free DMEM. A JPK NanoWizard-II Atomic Force Microscope (AFM) with BioCell (JPK Instruments, Berlin, Germany) was equipped to detect the nuclear stiffness of cell clones as previously described [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Cell topology study was carried out by scanning over areas of 100\u0026times;100 \u0026micro;m in contact mode using an FV1000 confocal microscope (Olympus, Shinjuku, Japan). The average cell height was calculated from 15 cells per stable clone and presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e\n\u003ch3\u003eDetermination of DNA content loss\u003c/h3\u003e\n\u003cp\u003eCells were stained with cell-permeable DAPI (#268298; Sigma-Aldrich, Darmstadt, Germany) and investigated under an FV1000 confocal microscope. The nuclear volume and DNA content were calculated using DAPI images generated by sequential z-series scans. Cavitation of the cell nucleus was visualized based on the DAPI intensity. Alternatively, cells were stained with a mixture of RNase (10 \u0026micro;g/mL) and propidium iodide (50 \u0026micro;g/mL), and the DNA content (at 617 nm) was analyzed using an LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ). Additionally, a comet assay was performed to detect DNA breaks (ab238544; Abcam).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining and imaging\u003c/h3\u003e\n\u003cp\u003eCells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. After blocking (10% FBS, 0.3% Triton X-100, 100mM Tris, 150mM NaCl, pH\u0026thinsp;=\u0026thinsp;7.5) for 1-hr, the cells were incubated with primary antibodies (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) at 4\u0026deg;C overnight. On the second day, the cells were washed and stained with secondary antibodies for 1-hr. For actin staining, cells were washed with warm PBS, fixed, and stained with Rhodamine-conjugated phalloidin (R415; Thermo Fisher Scientific). NIR-Cytopainter (ab176833; Abcam, Cambridge, UK), was used for mitochondrial staining. The cells were counterstained with DAPI and visualized under an IX83 fluorescence microscope (Olympus).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eActin alignment score (AAS)\u003c/h2\u003e\u003cp\u003eThe angular distribution of stress fibers was analyzed using AAS, a parameter modified from a previous study [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In brief, the images of each stress fiber (length\u0026thinsp;\u0026gt;\u0026thinsp;10\u0026micro;m) were individually extracted. Filament angles to migration direction were estimated using CDC42 as the polarization marker, combined with the largest cell length. Then, every angle was converted into its corresponding Tensor, where Tensor\u0026thinsp;=\u0026thinsp;RADIANS(filament angle \u0026ndash; the average angle in one cell). AAS\u0026thinsp;=\u0026thinsp;100\u0026times;Cos(radius). The average AAS was calculated from 15 cells per stable clone and presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell-based functional studies\u003c/h3\u003e\n\u003cp\u003eCell proliferation was assessed by MTT assay. For 3D co-culture study, 10\u003csup\u003e4\u003c/sup\u003e mCherry-expressing CAL27 cells were mixed with equal amounts of cells expressing different constructs and seeded into a hanging drop plate (3D Biomatrix, Ann Arbor, MI). Cell images were taken after 10-days and the sphere sizes were analyzed using Image-Pro Premier. To assess cell stemness, limiting dilution analysis was performed by seeding cells in 96-well ultra-low attachment plates (Corning, Avon, France) at densities of 1, 10, 100, or 1000 cells/well. After 10-days, visible spheres (diameter\u0026thinsp;\u0026gt;\u0026thinsp;20\u0026micro;m) were counted in each well. The sphere formation activity was calculated using ELDA (bioinf.wehi.edu.au/software/elda/) and quantified as scores for sphere-forming efficiency (SFE) and cancer-initiating cells (CIC).\u003c/p\u003e\u003cp\u003eCell invasiveness was assessed using Boyden chamber (Merck Millipore). Additionally, THUNDER live cell system (Leica Microsystems, Wetzlar, Germany) was utilized to decode the invadopodia of cells grown on collagen, labeled with Alexa-Fluo-568 NHS Ester (A20103; Thermo Fisher Scientific). To study stroma-dependent cell growth, 3\u0026times;10\u003csup\u003e5\u003c/sup\u003e HOrFs were seeded on NaOH-pretreated coverslips in 24-well plates. After 48-hr, 10\u003csup\u003e5\u003c/sup\u003e Cal27 cells per well were seeded on top of the HOrFs and cultured for 3-days in the presence or absence of 1 \u0026micro;M FGFR inhibitors. Cells on the coverslips were fixed and stained using a Masson Tricolor Staining Kit (G1006; Servicebio, Wuhan, China).\u003c/p\u003e\n\u003ch3\u003eTranscriptome analysis\u003c/h3\u003e\n\u003cp\u003eRNA samples were extracted from cells transiently or constitutively expressing gene constructs using an RNeasy Kit (Qiagen, Hilden, Germany). To study intercellular communication, RNA samples were also prepared from HOrFs treated with conditioned media for 48-hr. Samples from transiently transfected cells were sent for microarray analysis (Clariom_D_Human) at the Genomics Center, Kaohsiung Chang-Gung Memorial Hospital, Taiwan. RNA samples from the stable cell clones or educated HOrFs were sent for RNA sequencing (RNA-seq) service at Biotools (Taipei, Taiwan), utilizing NovaSeq 6000 platform (Illumina, San Diego, CA). Gene-set enrichment analysis (GSEA) was performed on the processed microarray or RNA-seq data using GSEA Java (version-4.3.3) from the Broad Institute. The functional interactome of upregulated and downregulated genes was analyzed using STRING protein-protein interaction database (string-db.org/). TOPCONS (topcons.cbr.su.se/) was utilized to define secreted or membrane proteins [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and CellChatDB dataset (cellchat.org/cellchatdb/) was utilized to clarify the intercellular communications [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eUltrastructural analysis by transmission electron microscope (tEM)\u003c/h2\u003e\u003cp\u003eUnless specified, all chemicals in this study were purchased from Sigma-Aldrich. Cells were cultured in suspension (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells) in 10 cm Petri dishes. After 48-hr, the spheroids were collected by centrifugation, followed by fixation with 2.5% glutaraldehyde (G5882) in 0.1M cacodylate buffer (C4946; pH 7.4) for overnight at 4\u0026deg;C. After washes, the spheroids were immersed with 1% OsO\u003csub\u003e4\u003c/sub\u003e (#201030) for 2-hr at 4\u0026deg;C and stained with UA zero EM (R1000; Agar Scientific, Essex, UK) for 1-hr. After washes with nanopure water, cells were dehydrated in graded ethanol solutions from 50\u0026ndash;100%. An epoxy-embedding kit (#45359) was used to embed the samples in beem capsules, then cured at 45\u0026deg;C for 24-hr and 60\u0026deg;C for 48-hr. Sections (50\u0026ndash;70 nm) were prepared and placed on 100 mesh copper grids for image observation using an HT7500 tEM (Hitachi High-Tech, Tokyo, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eTumor xenografts in nude mice\u003c/h2\u003e\u003cp\u003eSix-week-old male athymic \u003cem\u003enu/nu\u003c/em\u003e mice were purchased from the National Laboratory Animal Center, Taiwan and randomly assigned into groups. Stable cell clones were mixed with matrigel (1:1 v/v) (DLW356231; Sigma-Aldrich) and injected subcutaneously into mice (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells per injection, two sites per mouse, 5 mice per group). Sixty-days after cell inoculation or when the tumor size reached 3000 mm\u0026sup3;, the mice were euthanized. Tissue sections were prepared for histological examination using previously described protocols [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. All studies were conducted under the guidelines of the Institutional Animal Care and Use Committee at National Sun Yatsen University (IACUC-10909 and \u0026minus;\u0026thinsp;11126).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA) and SPSS (version-14.0; SPSS Inc., Chicago, IL). A \u003cem\u003et\u003c/em\u003e-test or \u003cem\u003eFisher\u0026rsquo;s\u003c/em\u003e exact test was used for comparisons between two groups, whereas one-way ANOVA was used for multiple group comparisons. A \u003cem\u003ep\u003c/em\u003e-value significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eAcute K6-K14/V5 expression triggers nuclear deformation and DNA breaks, leading to cGAS-STING activation\u003c/h2\u003e\u003cp\u003eWe have previously identified seven major K6-K14 keratin fusions in HNSCC [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among them, the K6-K14/V5 variant tends to form aggregates prominently around the cell nucleus, resulting in the complete loss of wild-type keratin network within cells. To investigate its functional roles in cancer development, gene constructs were expressed in Cal27 and FaDu cells which show undetectable fusion events (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Dislike clear IF network formed by K14, K6-K14/V5 expression led to aggregates, accompanied by higher frequencies of nuclear deformation and micronuclei in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). By co-transfecting mCherry-tagged SUN2, a key component of the LINC complex, K6-K14/V5 expression showed significant damage to the nuclei (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). Cell topography revealed that K6-K14/V5 exerted unbalanced mechanical forces, leading to nuclear distortion and the formation of hollow nuclei (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB, Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). The DNA content was reduced accordingly (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB) with a notable reduction in nuclear volume (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD) and stiffness (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eE). Since DNA damage can cause centrosome disorganization [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], K6-K14/V5 was found to form aggregates with centrosomes with increased total number (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eComet assay clearly detected DNA breaks in cells expressing K6-K14/V5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), associated with the formation of phospho-CHK2 (p-CHK2) and γH2AX foci in the nuclei in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These findings suggest the activation of ATM/ATR-mediated DDRs via the p53-p21 cascade, as verified by western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Increased levels of γH2AX ubiquitination, a marker for active induction of DNA lesions [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], were also observed. Cell proliferation was significantly suppressed which can be partially rescued by an ATM/ATR inhibitor CGK733 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Among the 12 upregulated reactomes (genes with fold change\u0026thinsp;\u0026gt;\u0026thinsp;2.0 compared to K14 control), three indicated the involvement of cytosolic DNA-induced IFN signaling (Figure S3). Immunostaining further confirmed the activation of cGAS-STING signaling associated with the DDRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eAcute K6-K14/V5 expression reduces mechanosensing and alters the preference for matrix rigidity\u003c/h2\u003e\u003cp\u003eThe LINC complex is a central component of mechanosensing, thus K6-K14/V5 expression might influence the mechanical properties. F-actin staining revealed well-organized networks in the control cells, enabling cell extension and spreading. In contrast, less cell protrusion with reduced focal adhesion formation and spreading area was observed in cells acutely expressing K6-K14/V5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting less intracellular tension and force transmission [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. With reduced cortical actomyosin contractility, topological study by atomic force microscope (AFM) confirmed increased cell height in cells expressing K6-K14/V5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Confocal microscopy further revealed relaxed cell nuclei with expansion in the z-direction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Western blotting indicated the downregulation of mechanosensing, including focal adhesion-mediated integrin signaling (p-ITGB1, p-FAK, p-PI3K, and p-MAPK1) and intracellular contractility (PAK1 and myosin IIb) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These findings are very similar to those reported in a previous study that disruption of keratin stability by overexpressing a dominant K14 mutation R416P inhibited the normal mechanical response to substrate rigidity, reducing F-actin stress fibers and cell stiffness [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBecause changes in rigidity sensing and matrix preference is a mechanical phenotype during cell transformation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], we compared the cell growth ability by seeding K6-K14/V5-expressing cells on regular culture dishes or dishes covered with collagen gels under G418 selection. As expected, K6-K14/V5-positive cells lost their population after several passages on regular dishes, whereas the same transfected cells could survive on the collagen and finally became the major population, named as 3D-V5 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Strikingly, the surviving 3D-V5 cells also changed their morphology after two weeks and formed sphere-like aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eInduction of cancer dormancy via autophagy in the surviving 3D-V5 cells\u003c/h2\u003e\u003cp\u003eNotably, the surviving 3D-V5 cells constitutively activated IFN-mediated signaling (Figure S4) and did not abate cGAS-STING signaling as previously proposed [5. 6], suggesting the existence of other mechanisms to subvert lethal epithelial responses to cytosolic DNA [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For this, gene-set enrichment analysis (GSEA) revealed some enriched pathways contributing to cancer dormancy, including downregulation of cell cycle progression, focal adhesion-mediated signaling, ERK/MAPK signaling, and immunogenic potentials (Figure S4). In line with these, upregulation of drug resistance and retinoic acid signaling were also observed. Several studies have shown autophagy as a key mechanism for cancer cells to overcome cellular stresses via dormancy induction [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In our study, several genes involved in autophagic process were found upregulated in 3D-V5 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), which can be confirmed by immunostaining and western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Ultrastructural analysis also revealed an increase in mitochondrial fission, resulting in elevated small mitochondria with some in autophagic vacuoles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Upregulation of metabolic reprogramming processes associated with autophagy-induced cell dormancy was also found in 3D-V5 cells (Figure S4). Among them, pathways contributing to vitamin A-retinoic acid signaling were most significantly activated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), which regulates stem cell dormancy and cellular plasticity via remodeling spatial chromatin context [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eLong-term K6-K14/V5 expression reprograms keratin expression profiles with a partial-EMT (pEMT) phenotype\u003c/h2\u003e\u003cp\u003eConsidering DDR-induced catastrophe with a self-defined fitness may serve as selective pressure for cancer evolution [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], we then determined whether the surviving 3D-V5 cells show different phenotypes from those with acute K6-K14/V5 expression. First, 3D-V5 cells could survive on a regular culture dish and partially recover the IF network with visible nuclear array. The LINC complex maintained in a compact shape, although some were empty without detectable DNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). GSEA revealed keratinization as the most upregulated pathway in 3D-V5 cells as compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cem\u003eupper\u003c/em\u003e; Figure S4), indicating the assistance of additional keratin members to reorganize K6-K14/V5 network in the surviving cells. Several altered keratins have been linked to an aggressive program for epithelial tumors, including upregulation of K19, K14, K15, K6, and K17, as well as downregulation of K8/K18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cem\u003elower\u003c/em\u003e) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine the stemness potential, we screened the expression levels of gene signatures for epithelial (E) and mesenchymal (M) phenotypes in HNSCC [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. 3D-V5 cells tended to gain a higher E-score than control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), however, they did not show increased levels of CDH1 and EPCAM. In contrast, certain mesenchymal genes were upregulated (e.g., EMILIN1, LAMA4, FOXC2, MMP2, and ITGA5), whereas an overall lower M-score was found. Those data suggest a transition state between full-E and full-M programs. To confirm this, we analyzed gene signatures for pEMT program in HNSCC [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. K14-expressing cells showed a higher pEMT score than GFP cells as previously proposed [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Nevertheless, 3D-V5 cells harbored an even higher pEMT potential with extremely high levels (more than 2\u003csup\u003e6\u003c/sup\u003e folds) of MMP10, ACKR3 (CXCR7), and SOX2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eWhen co-cultured with mCherry-expressing parental cells in hanging drops, 3D-V5 cells localized to the surface of cell spheres and generated a unique microenvironment to nurture the sphere growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), a potent feature for HNSCC cells at pEMT state [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although K14-expressing cells promoted sphere growth, they did not form a clear \u0026ldquo;cell shell\u0026rdquo; outside the sphere. Through limiting dilution assay, increased stemness and plasticity were confirmed in 3D-V5 cells (SFE: 24.5% and CIC: 1/8.2), as compared to K14- (SFE: 12.3% and CIC: 1/51.0) and GFP-expressing (SFE: 6.5% and CIC: 1/80.9) cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These data suggest that long-term expression and selection by K6-K14/V5 promotes cancer aggressiveness by upregulating pEMT program through keratin network reorganization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eSurviving 3D-V5 cells regain mechanical strength to promote cell protrusion and invasion\u003c/h2\u003e\u003cp\u003eIn addition to oncogenic keratins, several genes related to actin and microtubule networks were also upregulated, which form compact interactomes for cytoskeleton organization and actin-mediated cell contraction, suggesting the recovery of mechanical properties in the surviving cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; Table S3). Consistently, the nuclear stiffness of 3D-V5 cells was found to increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). As previously shown, overexpression of Piezo2 mechanosensitive channel is essential to enhance mechano/biological responses involved in cell-matrix sensing and cellular adaptation in the tumor microenvironment (TME) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which could also be detected in 3D-V5 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The downstream effectors of Pieozo2-mediated Ca\u003csup\u003e2+\u003c/sup\u003e-influx were also upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Actin filament staining revealed increased stress fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and a higher alignment value (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) in 3D-V5 cells, suggesting the strengthening ability to migrate and invade [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. When grown in a 3D microenvironment, 3D-V5 cells, especially those on the surface layer of the spheres, showed striking cell protrusions, supporting the pEMT phenotype for active interactions with the surrounding ECM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Additionally, data from both transcriptome and protein array studies confirmed upregulation of MMP1, MMP10, and MMP13 in 3D-V5 cells, which associated with the downregulation of their inhibitor, TIMP-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). When cultured on collagen gel, enhanced invadipodia protrusion was found in 3D-V5 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Transwell assay further indicated a significantly increased ECM-invasion ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). These data suggest enhanced mechanosensing and invasiveness in 3D-V5 cells via cytoskeleton reorganization.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eSurviving 3D-V5 cells promote tumor formation via extracellular vesicle (EV)-mediated intercellular interactions\u003c/h2\u003e\u003cp\u003eWhen observed under tEM, we noticed elevated EVs secreted from 3D-V5 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). EV isolation and quantification further confirmed this finding (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), suggesting active intercellular interactions. As compared to control cells, more genes were upregulated in 3D-V5 cells, especially the ones annotated as membrane-bound or secreted proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. When culturing human oral fibroblasts (HOrFs) with conditioned media, more expression alterations were found in 3D-V5-educated HOrFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Through ligand-receptor pairing [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], the major intercellular communications were mediated by those related to tumor growth, cancer-associated fibroblast (CAF) activation/proliferation, ECM remodeling, drug resistance, and neuron axon guidance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC; Figure S5). Notably, PLXNA4 was overexpressed in treated HOrFs, and the expression of its receptor, SEMA6A, was enhanced in both 3D-V5 cells and treated HOrFs. Since SEMA6A also acts as a ligand for PLXNA4, the PLXNA4-SEMA6A interaction can promote cell activation/proliferation in both cancer cells and CAFs [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Overexpression of ERBB2, FGFR1, and FGFR3 were also found in 3D-V5 cells, which are known to be co-activated by SEMA6A [32. 33]. Moreover, 3D-V5-educated HOrFs showed increased production of FGF10, FGF17, KLRD1, and CXCL12, contributing to FGFR1/FGFR3 signaling activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC; Figure S5). FGF13 and FGF22 were also overexpressed in 3D-V5 cells, serving as autocrine/paracrine factors to enhance FGFR signaling. Consistently, 3D-V5 cells exhibited a significantly higher proliferation rate than control cells when co-cultured with HOrFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), which could be partially suppressed by selective FGFR inhibitors, BGJ-398 and AZD4547 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn a mouse xenograft model, GFP-expressing cells formed well-differentiated lesions with clear keratin pearls, and K14-expressing cells generated moderately differentiated lesions with viable cancer cells arranged in the nests (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Dramatically, 3D-V5 lesions showed a poorly-differentiated phenotype, exhibiting variations in cell size and shape along with an infiltrative pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Especially, cancer cells in the center of the lesion show active cannibalistic activity, which is frequently coupled with extracellular acidosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This finding suggests that degradation of the \u0026ldquo;loser\u0026rdquo; cells in acidic conditions could provide sufficient nutrients to nurture the \u0026ldquo;winner\u0026rdquo; cells with higher cancer stemness (CD44\u003csup\u003e+\u003c/sup\u003e) and proliferation rate (Ki-67\u003csup\u003e+\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Intense CD44 staining was also detected at the invasive front of 3D-V5 lesions, suggesting active interactions between \u0026ldquo;winner\u0026rdquo; cells and the ECM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Approximately 40% (4 out of 10) of 3D-V5 lesions showed high CD44 expression and invasiveness to metastasize from subcutaneous layer into peritoneal cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Strong FGFR1/FGFR3 activation can be detected the interface between tumor lesions and the ECM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), guiding 3D-V5 metastasis.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the molecular complexity of keratin IF networks in cancer cells and their impact on cellular adaptation and clonal selection/evolution following a nuclear catastrophe induced by K6-K14/V5 expression. By promoting autophagy, K6-K14/V5-expressing cancer cells with high genome instability can survive on collagen gel in a pro-dormant state, while altering their transcriptional profiles. As a result, the surviving cancer cells exhibit enhanced epithelial-mesenchymal dynamics and acquire cancer stemness. Since IFs are key components of the cellular architecture, K6-K14/V5-induced IF network disruption and the subsequent cytoskeleton reorganization provide cancer cells with elevated plasticity, allowing them to adapt to diverse ECM. A complex tumor ecosystem with active crosstalk between cancer and stromal cells can be established, ultimately leading to the reactivation of cancer dormancy and tumor spread. Our study presents a novel model for exploring the impact of changes in the mechanical properties of cells on cancer plasticity and complexity.\u003c/p\u003e\u003cp\u003eAutophagy can trigger cancer dormancy to escape dead signaling, alter gene expression profiles, and promote metabolic reprogramming, a survival strategy for cancer cells to adapt and regrow by genetic or epigenetic alterations through a Darwinian evolutionary process [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Based on this perspective, genome instability triggered by K6-K14/V5 expression may function as an accelerator to generate cell clones with high heterogeneity, allowing them to evolve greater adaptability. Significantly, mechanical cues via the interactions with ECM play both positive and negative roles in regulating autophagic processes by controlling integrin-mediated and YAP/TAZ signals [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In our study, elevated EVs were released from the surviving 3D-V5 cells, leading to the accumulation of a variety of FGF ligands, collagens, and proteoglycans in the ECM. This may establish a positive feedback loop in which more alterations in the ECM can enhance FGF/FGFR signaling [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], resulting in more active cellular responses in ECM remodeling. Whether such interactions between 3D-V5 cells and stromal cells can feedback control autophagic statuses of the cancer cells will be an interesting topic to explore.\u003c/p\u003e\u003cp\u003eFGF/FGFR signaling plays an important role in regulating cellular plasticity and promoting tumor recurrence; thus, it has been suggested as a potent target for treating patients with metastatic cancers [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Recent studies also support its involvement in HNSCC development and the therapeutic potential for patient treatment [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Nevertheless, the FGF-FGFR axis is just one of the key signaling pathways involved in the interactions between 3D-V5 cells and the ECM. Other factors contributing to CAF activation and dormancy reactivation need to be further defined. For examples, several matrix proteins, such as DCN, COL14A1, and COL15A1, were highly expressed in 3D-V5-educated HOrFs. These proteins have been reported to play dual roles in both tumor suppression and cancer progression, influencing the onset of cancer metastasis via ECM remodeling [\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Further studies are needed to explore how mechanical plasticity in cancer cells may contribute to ECM remodeling in controlling cancer dormancy reactivation.\u003c/p\u003e\u003cp\u003eA pEMT program at the leading edge of a tumor lesion has been suggested as an independent predictor of nodal metastasis, tumor grade, and adverse pathologic features in many cancer types, including HNSCC [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. With increased mechanical complexity, cancer cells can easily stretch out and form invasive tips, allowing cells to break away through gaps within tissues and start to metastasize [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In our study, invasive tips can be frequently detected in these cells both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e with higher migration and invasion ability in the surviving 3D-V5 cells with Piezo2 upregulation. In addition, 3D-V5 cells upregulated MMP1 and MMP10, which play roles in the collective detachment of cancer cells from the primary tumor site during distant metastasis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Notably, MMP10 is known to maintain a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in cancer lesions [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. These findings support the previous studies that dormant disseminated tumor cells share several similar phenotypes as cancer stem/progenitor-like cells [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. By promoting active cross-talk between cancer cells and the TME through FGFR signaling, dormant cancer cells can gain the ability to reactivate and metastasize.\u003c/p\u003e\u003cp\u003eThis study has some limitations. First, we did not clearly know how mechanosensing loss by acute k6-k14/V5 expression triggers cytoskeletal reorganization and transcriptome reprogramming in the surviving 3D-V5 cells. Transposase-accessible chromatin with sequencing (ATAC-seq) may provide a raw map of chromatin connections to address the changes in the spatial chromatin context. Since k6-k14/V5 expression can alter the preference for matrix rigidity, it is also necessary to explore the mechanical impact of matrix rigidity on cellular adaptation and the signaling balance between cancer dormancy and cell awakening. Finally, S100A8, S100A9, and other S100 Ca\u003csup\u003e2+\u003c/sup\u003e-binding proteins were upregulated in the surviving 3D-V5 cells (Table S4), which have been known as potent attractants for neutrophil recruitment/activation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Such immune editing can participate in different steps of tumorigenic processes, including cell motility/invasion/migration, cell cycle/cell proliferation, TME remodeling, drug resistance, and cancer stem cell formation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Whether 3D-V5 directly influences neutrophil activity to facilitate cancer metastasis requires further investigation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData from the experiments presented in this study are included in this published article and its Supplementary Information files. The transcriptome sequencing data are accessible at Gene Expression Omnibus (GEO) Databank (GSE292626).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors thank the technical assistance from Ms. Ting-Chia Chiang at National Sun Yatsen University/Taiwan. The authors also like to thank critical comments and suggestions from Prof. Tian-Li Wang at the John Hopkins Medical Institutions/USA, Prof. Dennis E. Discher at University of Pennsylvania/USA, and Prof. Chih-Wen Shu at National Sun Yatsen University/Taiwan. This study was supported by grants from National Science and Technology Council, Taiwan (112-2320-B-110-005-MY3, 109-2314-B-110-003-MY3, 113-2811-B-110-007), Kaohsiung Veterans General Hospital, Taiwan (KSVGH-113-101, KSVGH112-079), and Ministry of Health and Welfare, Taiwan (11126, 10916).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIHC:\u003c/strong\u003e conceptualization, investigation, writing\u0026mdash;original draft, writing\u0026mdash;review \u0026amp; editing, and funding acquisition. \u003cstrong\u003eSR:\u003c/strong\u003e methodology, investigation, formal analysis, visualization, writing\u0026mdash;original draft, and writing\u0026mdash;review \u0026amp; editing. \u003cstrong\u003eMTL:\u003c/strong\u003e conceptualization, investigation, writing\u0026mdash;original draft, and writing\u0026mdash;review \u0026amp; editing. \u003cstrong\u003eCCY:\u003c/strong\u003e conceptualization, supervision, and funding acquisition. \u003cstrong\u003eBYTK:\u003c/strong\u003e methodology, investigation, and formal analysis. \u003cstrong\u003eLWC:\u003c/strong\u003e investigation and formal analysis. \u003cstrong\u003eJY:\u003c/strong\u003e methodology and investigation. \u003cstrong\u003eWLW:\u003c/strong\u003e investigation and visualization. \u003cstrong\u003eKS:\u0026nbsp;\u003c/strong\u003einvestigation and formal analysis. \u003cstrong\u003eHHC:\u003c/strong\u003e investigation and formal analysis. \u003cstrong\u003eTH:\u003c/strong\u003e visualization. \u003cstrong\u003eCMC:\u003c/strong\u003e visualization. \u003cstrong\u003eIHS:\u003c/strong\u003e methodology and supervision. \u003cstrong\u003eMJT:\u003c/strong\u003e methodology and supervision. \u003cstrong\u003eJJCS:\u003c/strong\u003e conceptualization, formal analysis, writing\u0026mdash;original draft, writing\u0026mdash;review \u0026amp; editing, and funding acquisition.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaraswathibhatla A, Indana D, Chaudhuri O. 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Biomed Pharmacother. 2020;127:110156.\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":"oncogenesis","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"oncsis","sideBox":"Learn more about [Oncogenesis](http://www.nature.com/oncsis/)","snPcode":"41389","submissionUrl":"https://mts-oncsis.nature.com/cgi-bin/main.plex","title":"Oncogenesis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cytoskeleton, cytokeratin, keratin fusion, cancer stemness, cellular plasticity, cGAS-STING, dormancy","lastPublishedDoi":"10.21203/rs.3.rs-6985645/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6985645/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCytoskeletal network dynamics play important roles in regulating cellular functions. Although alterations in cytoskeleton-related genes are frequently detected, limited attention has been paid to their roles in cancer development. A novel keratin fusion variant, K6-K14/V5, was previously identified in head and neck squamous cell carcinoma, and its expression led to catastrophic nuclear collapse, resulting in DNA breaks and cGAS-STING activation. Such cell-killing effects can trigger autophagy induction, which, in turn, promotes cancer cell evolution/clonal selection in a dormant state. Furthermore, due to the disrupted cellular architecture and the loss of mechanosensing, these dormant cells could only survive and adapt within a softer microenvironment, such as collagen gel. Upregulation of the partial epithelial-mesenchymal transition program by cytoskeleton reorganization was defined as a key step for these dormant cells to reactivate and regain their mechanical properties. Striking cell protrusions and increased MMPs were observed in the reactivated cells, facilitating interactions with the surrounding extracellular matrix and enhancing their invasive potential. Elevated extracellular vesicles were also found in the reactivated cells, which actively stimulated tumor growth via the FGF-FGFR axis. Our study offers a model for understanding how genetic alterations in cytoskeletal genes can directly contribute to cancer development and drive cancer evolution.\u003c/p\u003e","manuscriptTitle":"Cytoskeleton reorganization induced by a novel K6-K14 keratin fusion promotes cancer stemness and cellular plasticity via cGAS-STING selection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-24 18:29:35","doi":"10.21203/rs.3.rs-6985645/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-09-05T14:53:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-04T10:21:09+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-08-27T11:12:28+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-16T07:13:37+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-16T07:03:28+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-07-22T13:02:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-11T13:56:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogenesis","date":"2025-07-10T09:04:44+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-06-27T10:41:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-26T17:29:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogenesis","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"oncsis","sideBox":"Learn more about [Oncogenesis](http://www.nature.com/oncsis/)","snPcode":"41389","submissionUrl":"https://mts-oncsis.nature.com/cgi-bin/main.plex","title":"Oncogenesis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"22d4a8ca-238c-463a-9313-8ad86e2c836e","owner":[],"postedDate":"July 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51935003,"name":"Biological sciences/Cell biology/Cytoskeleton"},{"id":51935004,"name":"Biological sciences/Cancer/Oncogenes"}],"tags":[],"updatedAt":"2026-01-29T08:06:26+00:00","versionOfRecord":{"articleIdentity":"rs-6985645","link":"https://doi.org/10.1038/s41389-026-00598-1","journal":{"identity":"oncogenesis","isVorOnly":false,"title":"Oncogenesis"},"publishedOn":"2026-01-20 05:00:00","publishedOnDateReadable":"January 20th, 2026"},"versionCreatedAt":"2025-07-24 18:29:35","video":"","vorDoi":"10.1038/s41389-026-00598-1","vorDoiUrl":"https://doi.org/10.1038/s41389-026-00598-1","workflowStages":[]},"version":"v1","identity":"rs-6985645","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6985645","identity":"rs-6985645","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}