Spa2 remodels ADP-actin via molecular condensation under glucose starvation.

Actin nucleotide-dependent actin remodeling is essential to orchestrate signal transduction and cell adaptation. Rapid energy starvation requires accurate and timely reorganization of the actin network. Despite distinct treadmilling mechanisms of ADP- and ATP-actin filaments, their filament structures are nearly identical. How other actin-binding proteins regulate ADP-actin filament assembly is unclear. Here, we show that Spa2 which is the polarisome scaffold protein specifically remodels ADP-actin upon energy starvation in budding yeast. Spa2 triggers ADP-actin monomer nucleation rapidly through a dimeric core of Spa2 (aa 281-535). Concurrently, the intrinsically disordered region (IDR, aa 1-281) guides Spa2 undergoing phase separation and wetting on the surface of ADP-G-actin-derived F-actin and bundles the filaments. Both ADP-actin-specific nucleation and bundling activities of Spa2 are actin D-loop dependent. The IDR and nucleation core of Spa2 are evolutionarily conserved by coexistence in the fungus kingdom, suggesting a universal adaptation mechanism in the fungal kingdom in response to glucose starvation, regulating ADP-G-actin and ADP-F-actin with high nucleotide homogeneity.

Vasorin promotes endothelial differentiation of glioma stem cells via stimulating the transcription of VEGFR2.

Gliomas are highly vascularized malignancies, but current anti-angiogenic treatments have not demonstrated practical improvements in patient survival. Studies have suggested that glioma-derived endothelial cell (GdEC) formed by glioma stem cell (GSC) differentiation may contribute to the failure of this treatment. However, the molecular mechanisms involved in GSC endothelial differentiation remain poorly understood. We previously reported that vasorin (VASN) is highly expressed in glioma and promotes angiogenesis. Here, we show that VASN expression positively correlates with GdEC signatures in glioma patients. VASN promotes the endothelial differentiation capacity of GSC in vitro and participates in the formation of GSC-derived vessels in vivo. Mechanistically, vascular endothelial growth factor receptor 2 (VEGFR2) is a critical factor that mediates the regulation of VASN on GSC endothelial differentiation. Separation of cell chromatin fractionation and chromatin immunoprecipitation-sequencing analysis show that VASN interacts with Notch1 and co-translocates into the cell nuclei, where VASN binds to the VEGFR2 gene promoter to stimulate its transcription during the progression of GSC differentiation into GdEC. Together, these findings elucidate the role and mechanisms of VASN in promoting the endothelial differentiation of GSC and suggest VASN as a potential target for anti-angiogenic therapy based on intervention in GdEC formation in gliomas.

Detection of novel Plasmodium falciparum coronin gene mutations in a recrudescent ACT-treated patient in South-Western Nigeria.

Background: Routine surveillance for antimalarial drug resistance is critical to sustaining the efficacy of artemisinin-based Combination Therapies (ACTs). Plasmodium falciparum kelch-13 (Pfkelch-13) and non-Pfkelch-13 artemisinin (ART) resistance-associated mutations are uncommon in Africa. We investigated polymorphisms in Plasmodium falciparum actin-binding protein (Pfcoronin) associated with in vivo reduced sensitivity to ART in Nigeria. Methods: Fifty-two P. falciparum malaria subjects who met the inclusion criteria were followed up in a 28-day therapeutic efficacy study of artemether-lumefantrine in Lagos, Nigeria. Parasite detection was done by microscopy and molecular diagnostic approaches involving PCR amplification of genes for Pf18S rRNA, varATS, telomere-associated repetitive elements-2 (TARE-2). Pfcoronin and Pfkelch-13 genes were sequenced bi-directionally while clonality of infections was determined using 12 neutral P. falciparum microsatellite loci and msp2 analyses. Antimalarial drugs (sulfadoxine-pyrimethamine, amodiaquine, chloroquine and some quinolones) resistance variants (DHFR_51, DHFR_59, DHFR_108, DHFR_164, MDR1_86, MDR1_184, DHPS_581 and DHPS_613) were genotyped by high-resolution melting (HRM) analysis. Results: A total of 7 (26.92%) cases were identified either as early treatment failure, late parasitological failure or late clinical failure. Of the four post-treatment infections identified as recrudescence by msp2 genotypes, only one was classified as recrudescence by multilocus microsatellites genotyping. Microsatellite analysis revealed no significant difference in the mean allelic diversity, He, (P = 0.19, Mann-Whitney test). Allele sizes and frequency per locus implicated one isolate. Genetic analysis of this isolate identified two new Pfcoronin SNVs (I68G and L173F) in addition to the P76S earlier reported. Linkage-Disequilibrium as a standardized association index, IAS, between multiple P. falciparum loci revealed significant LD (IAS = 0.2865, P=0.02, Monte-Carlo simulation) around the neutral microsatellite loci. The pfdhfr/pfdhps/pfmdr1 drug resistance-associated haplotypes combinations, (108T/N/51I/164L/59R/581G/86Y/184F), were observed in two samples. Conclusion: Pfcoronin mutations identified in this study, with potential to impact parasite clearance, may guide investigations on emerging ART tolerance in Nigeria, and West African endemic countries.

The actin-binding protein CAP1 represses MRTF-SRF-dependent gene expression in mouse cerebral cortex.

Serum response factor (SRF) is an essential transcription factor for brain development and function. Here, we explored how an SRF cofactor, the actin monomer-sensing myocardin-related transcription factor MRTF, is regulated in mouse cortical neurons. We found that MRTF-dependent SRF activity in vitro and in vivo was repressed by cyclase-associated protein CAP1. Inactivation of the actin-binding protein CAP1 reduced the amount of actin monomers in the cytoplasm, which promoted nuclear MRTF translocation and MRTF-SRF activation. This function was independent of cofilin1 and actin-depolymerizing factor, and CAP1 loss of function in cortical neurons was not compensated by endogenous CAP2. Transcriptomic and proteomic analyses of cerebral cortex lysates from wild-type and Cap1 knockout mice supported the role of CAP1 in repressing MRTF-SRF-dependent signaling in vivo. Bioinformatic analysis identified likely MRTF-SRF target genes, which aligned with the transcriptomic and proteomic results. Together with our previous studies that implicated CAP1 in axonal growth cone function as well as the morphology and plasticity of excitatory synapses, our findings establish CAP1 as a crucial actin regulator in the brain relevant for formation of neuronal networks.

Deletion of IRE1α in podocytes exacerbates diabetic nephropathy in mice.

Protein misfolding in the endoplasmic reticulum (ER) of podocytes contributes to the pathogenesis of glomerular diseases. Protein misfolding activates the unfolded protein response (UPR), a compensatory signaling network. We address the role of the UPR and the UPR transducer, inositol-requiring enzyme 1α (IRE1α), in streptozotocin-induced diabetic nephropathy in mice. Diabetes caused progressive albuminuria in control mice that was exacerbated in podocyte-specific IRE1α knockout (KO) mice. Compared to diabetic controls, diabetic IRE1α KO mice showed reductions in podocyte number and synaptopodin. Glomerular ultrastructure was altered only in diabetic IRE1α KO mice; the major changes included widening of podocyte foot processes and glomerular basement membrane. Activation of the UPR and autophagy was evident in diabetic control, but not diabetic IRE1α KO mice. Analysis of human glomerular gene expression in the JuCKD-Glom database demonstrated induction of genes associated with the ER, UPR and autophagy in diabetic nephropathy. Thus, mice with podocyte-specific deletion of IRE1α demonstrate more severe diabetic nephropathy and attenuation of the glomerular UPR and autophagy, implying a protective effect of IRE1α. These results are consistent with data in human diabetic nephropathy and highlight the potential for therapeutically targeting these pathways.

A step-by-step guide to fragmenting bundled actin filaments.

There has long been conflicting evidence as to how bundled actin filaments, found in cellular structures such as filopodia, are disassembled. In this issue, Chikireddy et al. (https://doi.org/10.1083/jcb.202312106) provide a detailed in vitro analysis of the steps involved in fragmentation of fascin-bundled actin filaments and propose a novel mechanism for severing two-filament bundles.

A simple protocol to establish a conditionally immortalized mouse podocyte cell line.

Podocytes are specialized terminally differentiated cells in the glomerulus that are the primary target cells in many glomerular diseases. However, the current podocyte cell lines suffer from prolonged in vitro differentiation and limited survival time, which impede research progress. Therefore, it is necessary to establish a cell line that exhibits superior performance and characteristics. We propose a simple protocol to obtain an immortalized mouse podocyte cell (MPC) line from suckling mouse kidneys. Primary podocytes were cultured in vitro and infected with the SV40 tsA58 gene to obtain immortalized MPCs. The podocytes were characterized using Western blotting and quantitative real-time PCR. Podocyte injury was examined using the Cell Counting Kit-8 assay and flow cytometry. First, we successfully isolated an MPC line and identified 39 °C as the optimal differentiation temperature. Compared to undifferentiated MPCs, the expression of WT1 and synaptopodin was upregulated in differentiated MPCs. Second, the MPCs ceased proliferating at a nonpermissive temperature after day 4, and podocyte-specific proteins were expressed normally after at least 15 passages. Finally, podocyte injury models were induced to simulate podocyte injury in vitro. In summary, we provide a simple and popularized protocol to establish a conditionally immortalized MPC, which is a powerful tool for the study of podocytes.

Microtubule dependent sorting of actin-binding proteins in mitosis.

The patterns of Formin B and of the Arp2/3 complex formed during mitosis were studied in a mutant of Dictyostelium discoideum that produces multinucleate cells, which divide by the ingression of unilateral cleavage furrows. During cytokinesis the cells of this mutant remain spread on a glass surface where they generate a planar pattern based on the sorting-out of actin-binding proteins. During anaphase, Formin B and Arp2/3 became localized to the regions of microtubule asters around the centrosomes; Formin B in particular in the form of round, quite uniformly covered areas. These areas have been shown to be depleted of myosin II and the actin-filament crosslinker cortexillin, and to be avoided by cleavage furrows on their path into the cell.

The F-actin bundler SWAP-70 promotes tumor metastasis.

Dynamic rearrangements of the F-actin cytoskeleton are a hallmark of tumor metastasis. Thus, proteins that govern F-actin rearrangements are of major interest for understanding metastasis and potential therapies. We hypothesized that the unique F-actin binding and bundling protein SWAP-70 contributes importantly to metastasis. Orthotopic, ectopic, and short-term tail vein injection mouse breast and lung cancer models revealed a strong positive dependence of lung and bone metastasis on SWAP-70. Breast cancer cell growth, migration, adhesion, and invasion assays revealed SWAP-70's key role in these metastasis-related cell features and the requirement for SWAP-70 to bind F-actin. Biophysical experiments showed that tumor cell stiffness and deformability are negatively modulated by SWAP-70. Together, we present a hitherto undescribed, unique F-actin modulator as an important contributor to tumor metastasis.

Nuclear actin structure regulates chromatin accessibility.

Polymerized ß-actin may provide a structural basis for chromatin accessibility and actin transport into the nucleus can guide mesenchymal stem cell (MSC) differentiation. Using MSC, we show that using CK666 to inhibit Arp2/3 directed secondary actin branching results in decreased nuclear actin structure, and significantly alters chromatin access measured with ATACseq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which enhances nuclear actin structure. In addition, nuclear visualization shows Arp2/3 inhibition decreases pericentric H3K9me3 marks. CytoD, alternatively, induces redistribution of H3K27me3 marks centrally. Such alterations in chromatin landscape are consistent with differential gene expression associated with distinctive differentiation patterns. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest increase in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions.

CARMIL1 regulates liver cancer cell proliferation by activating the ERK/mTOR pathway through the TRIM27/p53 axis.

Capping protein regulatory factor and myosin 1 linker 1 is termed CARMIL1. CARMIL1 is involved in several physiological processes; it forms an actin filament network and plasma membrane-bound cellular projection tissues and positively regulates the cellular components and tissues. CARMIL1 exhibits important biological functions in cancer; nonetheless, these functions have not been completely explored. We aimed to investigate the novel functions of CARMIL1 in liver cancer, particularly in cell proliferation. The cell counting kit-8, 5-ethynyl-2'-deoxyuridine, Component A experiments, and subcutaneous tumor formation model suggest that CARMIL1 is central to the proliferation of liver cancer cells both in vivo and in vitro. We extracted CARMIL1 samples from The Cancer Genome Atlas Program and analyzed its enrichment. CARMIL1 regulated the pathway activity by affecting the expression of star molecular proteins of the extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR). Moreover, it influenced the proliferation ability of liver cancer cells. Western blotting suggested that CARMIL1 downregulation could affect ERK and mTOR phosphorylation. Results of the co-immunoprecipitation demonstrated that CARMIL1 binds to tripartite motif (TRIM)27, which in turn binds to p53. Subsequently, CARMIL1 can regulate p53 stability and promote its degradation through TRIM27. Additionally, CARMIL1 inhibition enhanced the sensitivity of liver cancer cells to sorafenib. Tumor growth was significantly inhibited in the group treated with sorafenib and CARMIL1, compared with the group treated with CARMIL1 alone. Sorafenib is a first-line targeted chemotherapeutic drug for hepatocellular carcinoma treatment. It increases the long-term survival of hepatocellular carcinoma by 44%. In this study, downregulated CARMIL1 combined with sorafenib significantly reduced the tumor volume and weight of the mouse subcutaneous tumor model, indicating the potential possibility of combining CARMIL1 with sorafenib in hepatocellular carcinoma treatment. In summary, CARMIL1 promotes liver cancer cell proliferation by regulating the TRIM27/p53 axis and activating the ERK/mTOR pathway.

The dysadherin/FAK axis promotes individual cell migration in colon cancer.

Dysregulation of cancer cell motility is a key driver of invasion and metastasis. High dysadherin expression in cancer cells is correlated with invasion and metastasis. Here, we found the molecular mechanism by which dysadherin regulates the migration and invasion of colon cancer (CC). Comprehensive analysis using single-cell RNA sequencing data from CC patients revealed that high dysadherin expression in cells is linked to cell migration-related gene signatures. We confirmed that the deletion of dysadherin in tumor cells hindered local invasion and distant migration using in vivo tumor models. In this context, by performing cell morphological analysis, we found that aberrant cell migration resulted from impaired actin dynamics, focal adhesion turnover and protrusive structure formation upon dysadherin expression. Mechanistically, the activation of focal adhesion kinase (FAK) was observed in dysadherin-enriched cells. The dysadherin/FAK axis enhanced cell migration and invasion by activating the FAK downstream cascade, which includes the Rho family of small GTPases. Overall, this study illuminates the role of dysadherin in modulating cancer cell migration by forcing actin dynamics and protrusive structure formation via FAK signaling, indicating that targeting dysadherin may be a potential therapeutic strategy for CC patients.

Human disease-causing mutations result in loss of leiomodin 2 through nonsense-mediated mRNA decay.

The leiomodin (Lmod) family of actin-binding proteins play a critical role in muscle function, highlighted by the fact that mutations in all three family members (LMOD1-3) result in human myopathies. Mutations in the cardiac predominant isoform, LMOD2 lead to severe neonatal dilated cardiomyopathy. Most of the disease-causing mutations in the LMOD gene family are nonsense, or frameshift, mutations predicted to result in expression of truncated proteins. However, in nearly all cases of disease, little to no LMOD protein is expressed. We show here that nonsense-mediated mRNA decay, a cellular mechanism which eliminates mRNAs with premature termination codons, underlies loss of mutant protein from two independent LMOD2 disease-causing mutations. Furthermore, we generated steric-blocking oligonucleotides that obstruct deposition of the exon junction complex, preventing nonsense-mediated mRNA decay of mutant LMOD2 transcripts, thereby restoring mutant protein expression. Our investigation lays the initial groundwork for potential therapeutic intervention in LMOD-linked myopathies.

KLF13 promotes VSMCs phenotypic dedifferentiation by directly binding to the SM22α promoter.

Krüppel-like factor 13 (KLF13), a zinc finger transcription factor, is considered as a potential regulator of cardiomyocyte differentiation and proliferation during heart morphogenesis. However, its precise role in the dedifferentiation of vascular smooth muscle cells (VSMCs) during atherosclerosis and neointimal formation after injury remains poorly understood. In this study, we investigated the relationship between KLF13 and SM22α expression in normal and atherosclerotic plaques by bioanalysis, and observed a significant increase in KLF13 levels in the atherosclerotic plaques of both human patients and ApoE-/- mice. Knockdown of KLF13 was found to ameliorate intimal hyperplasia following carotid artery injury. Furthermore, we discovered that KLF13 directly binds to the SM22α promoter, leading to the phenotypic dedifferentiation of VSMCs. Remarkably, we observed a significant inhibition of platelet-derived growth factor BB-induced VSMCs dedifferentiation, proliferation, and migration when knocked down KLF13 in VSMCs. This inhibitory effect of KLF13 knockdown on VCMC function was, at least in part, mediated by the inactivation of p-AKT signaling in VSMCs. Overall, our findings shed light on a potential therapeutic target for treating atherosclerotic lesions and restenosis after vascular injury.

Kinetic trapping organizes actin filaments within liquid-like protein droplets.

Several actin-binding proteins (ABPs) phase separate to form condensates capable of curating the actin network shapes. Here, we use computational modeling to understand the principles of actin network organization within VASP condensate droplets. Our simulations reveal that the different actin shapes, namely shells, rings, and mixture states are highly dependent on the kinetics of VASP-actin interactions, suggesting that they arise from kinetic trapping. Specifically, we show that reducing the residence time of VASP on actin filaments reduces degree of bundling, thereby promoting assembly of shells rather than rings. We validate the model predictions experimentally using a VASP-mutant with decreased bundling capability. Finally, we investigate the ring opening within deformed droplets and found that the sphere-to-ellipsoid transition is favored under a wide range of filament lengths while the ellipsoid-to-rod transition is only permitted when filaments have a specific range of lengths. Our findings highlight key mechanisms of actin organization within phase-separated ABPs.

The roles of yeast formins and their regulators Bud6 and Bil2 in the pheromone response.

In response to pheromone Saccharomyces cerevisiae extend a mating projection. This process depends on the formation of polarized actin cables which direct secretion to the mating tip and translocate the nucleus for karyogamy. Here, we demonstrate that proper mating projection formation requires the formin Bni1, as well as the actin nucleation promoting activities of Bud6, but not the formin Bnr1. Further, Bni1 is required for pheromone gradient tracking. Our work also reveals unexpected new functions for Bil2 in the pheromone response. Previously we identified Bil2 as a direct inhibitor of Bnr1 during vegetative cell growth. Here, we show that Bil2 has Bnr1-independent functions in spatially focusing Bni1-GFP at mating projection tips, and in vitro Bil2 and its binding partner Bud6 organize Bni1 into clusters that nucleate actin assembly. bil2∆ cells also display entangled Bni1-generated actin cable arrays and defects in secretory vesicle transport and nuclear positioning. At low pheromone concentrations, bil2∆ cells are delayed in establishing a polarity axis, and at high concentrations they prematurely form a second and a third mating projection. Together, these results suggest that Bil2 promotes the proper formation and timing of mating projections by organizing Bni1 and maintaining a persistent axis of polarized growth.

Moesin deficiency leads to lupus-like nephritis with accumulation of CXCL13-producing patrolling monocytes.

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that link plasma membrane proteins to the cortical cytoskeleton and thus regulate diverse cellular processes. Mutations in the human moesin gene cause a primary immunodeficiency called X-linked moesin-associated immunodeficiency (X-MAID), which may be complicated by an autoimmune phenotype with kidney involvement. We previously reported that moesin-deficient mice exhibit lymphopenia similar to that of X-MAID and develop a lupus-like autoimmune phenotype with age. However, the mechanism through which moesin defects cause kidney pathology remains obscure. Here, we characterized immune cell infiltration and chemokine expression in the kidney of moesin-deficient mice. We found accumulation of CD4+ T and CD11b+ myeloid cells and high expression of CXCL13, whose upregulation was detected before the onset of overt nephritis. CD4+ T cell population contained IFN-γ-producing effectors and expressed the CXCL13 receptor CXCR5. Among myeloid cells, Ly6Clo patrolling monocytes and MHCIIlo macrophages markedly accumulated in moesin-deficient kidneys and expressed high CXCL13 levels, implicating the CXCL13-CXCR5 axis in nephritis development. Functionally, Ly6Clo monocytes from moesin-deficient mice showed reduced migration toward sphingosine 1-phosphate. These findings suggest that moesin plays a role in regulating patrolling monocyte homeostasis, and that its defects lead to nephritis associated with accumulation of CXCL13-producing monocytes and macrophages.

Restoring thalamocortical circuit dysfunction by correcting HCN channelopathy in Shank3 mutant mice.

Thalamocortical (TC) circuits are essential for sensory information processing. Clinical and preclinical studies of autism spectrum disorders (ASDs) have highlighted abnormal thalamic development and TC circuit dysfunction. However, mechanistic understanding of how TC dysfunction contributes to behavioral abnormalities in ASDs is limited. Here, our study on a Shank3 mouse model of ASD reveals TC neuron hyperexcitability with excessive burst firing and a temporal mismatch relationship with slow cortical rhythms during sleep. These TC electrophysiological alterations and the consequent sensory hypersensitivity and sleep fragmentation in Shank3 mutant mice are causally linked to HCN2 channelopathy. Restoring HCN2 function early in postnatal development via a viral approach or lamotrigine (LTG) ameliorates sensory and sleep problems. A retrospective case series also supports beneficial effects of LTG treatment on sensory behavior in ASD patients. Our study identifies a clinically relevant circuit mechanism and proposes a targeted molecular intervention for ASD-related behavioral impairments.

Nesprin-2 is a novel scaffold protein for telethonin and FHL-2 in the cardiomyocyte sarcomere.

Nesprins comprise a family of multi-isomeric scaffolding proteins, forming the linker of nucleoskeleton-and-cytoskeleton complex with lamin A/C, emerin and SUN1/2 at the nuclear envelope. Mutations in nesprin-1/-2 are associated with Emery-Dreifuss muscular dystrophy (EDMD) with conduction defects and dilated cardiomyopathy (DCM). We have previously observed sarcomeric staining of nesprin-1/-2 in cardiac and skeletal muscle, but nesprin function in this compartment remains unknown. In this study, we show that specific nesprin-2 isoforms are highly expressed in cardiac muscle and localize to the Z-disc and I band of the sarcomere. Expression of GFP-tagged nesprin-2 giant spectrin repeats 52 to 53, localized to the sarcomere of neonatal rat cardiomyocytes. Yeast two-hybrid screening of a cardiac muscle cDNA library identified telethonin and four-and-half LIM domain (FHL)-2 as potential nesprin-2 binding partners. GST pull-down and immunoprecipitation confirmed the individual interactions between nesprin-2/telethonin and nesprin-2/FHL-2, and showed that nesprin-2 and telethonin binding was dependent on telethonin phosphorylation status. Importantly, the interactions between these binding partners were impaired by mutations in nesprin-2, telethonin, and FHL-2 identified in EDMD with DCM and hypertrophic cardiomyopathy patients. These data suggest that nesprin-2 is a novel sarcomeric scaffold protein that may potentially participate in the maintenance and/or regulation of sarcomeric organization and function.

N6-methyladenosine methylation on FSCN1 mediated by METTL14/IGF2BP3 contributes to human papillomavirus type 16-infected cervical squamous cell carcinoma.

Human papillomavirus (HPV) infection has been reported to be associated with N6-methyladenosine (m6A) modification in cancers. However, the underlying mechanism by which m6A methylation participates in HPV-related cervical squamous cell carcinoma (CSCC) remains largely unclear. In this study, we observed that m6A regulators methyltransferase like protein (METTL14) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) were upregulated in HPV-positive CSCC tissues and cell lines, and their high expression predicted poor prognosis for HPV-infected CSCC patients. Cellular functional experiments verified that HPV16 oncogenes E6/E7 upregulated the expression of METTL14 and IGF2BP3 to promote cell proliferation and epithelial mesenchymal transition of CSCC cells. Next, we found that E6/E7 stabilized fascin actin-bundling protein 1 (FSCN1) mRNA and elevated FSCN1 expression in CSCC cells through upregulating METTL14/IGF2BP3-mediated m6A modification, and FSCN1 expression was also validated to be positively associated with worse outcomes of HPV-positive CSCC patients. Finally, HPV16-positive CSCC cell lines SiHa and CaSki were transfected with knockdown vector for E6/E7 or METTL14/IGF2BP3 and overexpressing vector for FSCN1, and functional verification experiments were performed through using MTT assay, flow cytometry, wound healing assay and tumour formation assay. Results indicated that knockdown of E6/E7 or METTL14/IGF2BP3 suppressed cell proliferation, migration and tumorigenesis, and accelerated cell apoptosis of HPV-positive CSCC cells. Their tumour-suppressive effects were abolished through overexpressing FSCN1. Overall, HPV E6/E7 advanced CSCC development through upregulating METTL14/IGF2BP3-mediated FSCN1 m6A modification.