Super-enhancer-driven ITGA6 enhances stemness features of squamous cell carcinoma through stabilizing c-myc protein

Super-enhancer-driven ITGA6 enhances stemness features of squamous cell carcinoma through stabilizing c-myc protein | 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 Super-enhancer-driven ITGA6 enhances stemness features of squamous cell carcinoma through stabilizing c-myc protein Bo Xiang, Ying Liu, Meng Hu, Jing Cai, Quanzhu Chen, Pan Chen, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3017766/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Squamous cell carcinoma (SCC) is life-threatening malignancy. Cancer stem cells (CSCs) are associated with SCCs initiation, metastasis, therapy resistance, and relapse. Acquisition of super enhancer is a cause of hyper-activation of oncogenes in cancer, contributing to tumorigenesis and progression. In this study, we found disruption of SEs-associated transcription by BRD4 inhibitors JQ1 and I-BET151 inhibited the stemness features of SCC cells. Combined analysis with transcriptomics alterations induced by treatments of BRD4 inhibitors and SEs profile of SCC cells identified SEs-driven oncogenes in SCC cells. Among these SEs-driven oncogenes, ITGA6 (Integrin subunit α6) was highly expressed in SCC samples and predicted unfavorable prognosis in SCC patients. The ΔNp63α, a lineage-survival oncogene in SCC, enriched at ITGA6-SEs and was responsible for the activation of ITGA6-SEs. Silencing of ITGA6 substantially impeded the stemness features in vitro, as well as reduced thenumber of tumor-initiating cells of SCC in vivo. Mechanistically, silencing of ITGA6 resulted in the degradation of c-Myc protein via upregulation of an E3 ubiquitin ligase FBXO32. Furthermore, we demonstrated silencing of ITGA6 promoted nuclear translocation of YAP1, which facilitated TEAD1-mediated transcription of FBXO32 in SCC cells. Thus, our data suggested ITGA6 contributes to maintaining stemness features of SCC through a YAP1/FBXO32/c-myc cascade, providing a therapeutic target for eliminating cancer stem cells. Biological sciences/Cancer/Cancer stem cells Biological sciences/Cancer/Head and neck cancer Integrin Cancer stem cells Super-enhancer ITGA6 Myc TP63 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Squamous cell carcinoma (SCC) is one of the most common solid neoplasms worldwide, mainly arising within the stratified epithelium of skin, lung, esophagus, aerodigestive or genitourinary tracts( 1 , 2 ). SCC is an aggressive malignancy, prone to metastasis, highly malignant, and has a poor prognosis, threatening human safety ( 3 , 4 ). However, the underlying mechanisms driving the malignant progression of SCC are not well understood. In the development of tumorigenesis, cancer stem cells (CSCs) are an important factor leading to tumor initiation, drug resistance, recurrence, and metastasis. Eliminating tumor stem cells is an effective treatment for tumors( 5 ). CSCs mainly originate from the accumulation of genetic mutations in primitive cells, activation of self-renewal programs, and inactivation of senescence and apoptosis programs ( 6 ). In addition, aberrant epigenetic alterations, including DNA methylation and histone modifications, play crucial roles in transforming normal stem cells to CSCs or the acquisition of stem-like features in cancer cells( 7 , 8 ). Enhancers emerge as a key noncoding cis-regulatory DNA sequence that can affect gene transcription. By acting on promoters, enhancers can regulate promoter activity and activate or enhance gene transcription( 9 , 10 ). Super enhancers (SEs) are large clusters of transcriptional enhancers that can play a prominent role in driving the expression of cell-type-specific genes through interacting with enriched transcription factors, co-factors, RNA polymerase II, and noncoding RNAs( 11 , 12 ). In human cancers, acquisition of SEs at oncogenes loci is an alternative mechanism for abnormal activation of oncogenes( 13 , 14 ). SEs are highly sensitive to the BRD4 inhibitor JQ1 and easily disturbed, so they can be a target for drug intervention( 15 ). SEs have been shown to affect the transcription of CSCs in SCC, thereby influencing the malignant behavior of the tumor ( 16 ). Thus, identifying SEs associated genes contributed to maintenance of CSCs characteristics may provide insights into the role of SEs in controlling stemness of CSCs and development of antitumor drugs. Integrins are the main cell adhesion receptors of extracellular matrix components, and they are a family of 24 transmembrane heterodimers generated from a combination of 18 α integrin and 8 β integrin subunits( 17 ). As a transmembrane signaling receptor, integrin can mediate epithelial cell adhesion, affect cell growth and differentiation, and is associated with tumor progression and poor prognosis in many epithelial malignant tumors( 18 , 19 ). Integrin α6 (ITGA6), also known as CD49f, is a member of the integrin family, which has been detected to be abnormally expressed in various tumors such as breast cancer, lung cancer, and liver cancer( 20 – 22 ). In this study, we demonstrated that disruption of SEs by BET inhibitors impaired the stemness features and malignant behaviors of SCC cells. We identified highly confident SEs associated genes in SCC cell line by integrative analysis H3K27ac ChIP-seq and RNA-seq following BET inhibitors treatment. Among these SEs associated genes, ITGA6 is highly expressed in various SCCs and associated with poor prognosis. Loss of ITGA6 accelerates proteasomal degradation of c-myc protein, leading to impairment of stemness features of SCCs cells. Inhibition of ITGA6 promotes nuclear translocation of YAP1 and activates the transcription of FBXO32, which acts as an E3 ligase targeting to c-myc protein. Methods Cell lines, cell culture and inhibitors HK1 is a highly differentiated squamous carcinoma cell from nasopharynx maintained in our lab( 23 ). BxPC3 is a pancreatic squamous carcinoma cell purchased from the National Infrastructure of Cell Line Resource (Shanghai, China)( 24 , 25 ). Cells were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, USA) and maintained in a humidified incubator consisting of 5% CO2 and 95% air at 37℃. JQ-1 (HY-13030) and I-BET151 (HY-13235) were purchased from MedChem Express (MCE, Monmouth Junction, USA). CCK8 assay CCK8 assay was performed according to formerly described( 26 ). Briefly, cells were seeded into 96-well plates at a density of 1×10 3 cells/100µL. Cells were allowed to grow for 0, 24, 48, 72, 96, and 120 h and were counted by using Cell Counting Kit (CCK-8). Besides, when cells were treated with drugs, 1×10 4 cells were seeded into 96-well plates supplemented 200µL culture medium containing the indicated dosage of JQ1 or I-BET151, then the cells were allowed to grow for 24 h and were measured by using CCK-8 assays. Colony formation assay Colony formation assay was performed as described previously( 27 ). HK1 and BxPC3 cells were seeded into 24-well plates at a density of 1 ×10 3 cells/well and cultured for about two weeks. When cells were treated with drugs, the original medium would be changed to a fresh complete medium containing the indicated dosage of JQ1 or I-BET151 after the cell in adherent. Finally, colonies were fixed with 4% paraformaldehyde and visualized with 1% crystal violet. These assays were performed in triplicate. Tumor sphere formation assay Tumor sphere formation assay was performed as previously described( 28 ). Briefly, 4×10 3 HK1 or BxPC3 cells were cultured in ultra-low adhesion six-well plates using sphere-forming medium-DMEM/F12 (Gibco) + EGF(20 ng/ml) (Gibco) + bFGF(10 ng/ml) (PeproTech) + B27(1×). Cells were treated with drugs by adding different concentrations of chemicals to the culture medium. After about 7–10 days, larger microspheres were observed and counted under a microscope. Cell migration and invasion assays Cell migration and invasion assays were performed as described previously( 29 , 30 ). 1×10 4 cells in serum-free medium were seeded into transwell inserts precoated with or without XXXatrigel. Then the inserts were put into the chamber of the 24-well plates which added with 700 µL culture medium containing 15% FBS. Then the transwells were incubated at 37℃ for 24-48h to allow tumor cells migrating or invading across the transwell membrane. The migrated or invaded tumor cells were then fixed with 4% paraformaldehyde and visualized with crystal violet. The number of migrated or invaded tumor cells were counted and photographed under a microscope. Atomic Force Microscopy (AFM) The biophysical properties of SCC cells were measured by AFM (JPK NanoWizard 4 BioScience, JPK Instruments, Germany) as described previously( 31 ). Briefly, cells were fixed in 2% glutaraldehyde for 45 s, 4% paraformaldehyde for 20 min, and then washed with PBS. AFM scanning were performed by using the probe HYDRA6V-100NG (AppNano, CA, USA) with a spring constant of 0.292 N/m. Cell adhesion, stiffness, and Rq were analyzed with JPK software. RNA extraction and real-time reverse-transcription PCR (RT-qPCR) Total intracellular RNA was extracted by using TRIzol Reagent (Life Technologies) as previously described( 32 ). For reverse transcription, 3 µg of total RNA samples were digested with DNase I (Takara, Beijing, China) to remove the trace amount of genomic DNA, then the RNA samples were reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kits (Thermo Fisher Scientific, Beijing, China). SYBR Green reagent (Bimake, Shanghai, China) was employed to perform Real-Time PCR assay by using CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Richmond, CA, USA). The 2 −ΔΔCT method was used to evaluate the relative expression levels of genes. PCR primers used in qPCR assays are listed in Table S1 . RNA-Seq RNA samples for RNA-Seq were prepared as described above, and sequencing was performed by an Illumina HiSeq platform (San Diego, CA, USA). The NOISeq method was employed to determine the differentially expressed genes with fold changes ≥ 2 ( 33 ). Gene set enrichment analysis (GSEA) was performed to analyze the result( 34 ). ChIP-qPCR Formaldehyde-cross-linked protein-DNA complexes were prepared from HK1, and BxPC3 live cells and cut into 200–1000 bp DNA fragments by ultrasound. Protein-DNA complexes were precipitated using anti-H3K27ac (active motif; Cat No. 39685), anti-TEAD1() antibody. The normal human immunoglobulin G (IgG) was used as negative control for antibody. The DNA fragments precipitated with antibodies were purified and determined by qPCR with specific Primers. Primers specific to each segment of interest are listed in Table S1 . Western blot analysis. Western Blot analysis was performed according to previously described( 35 ). Cells were lysed with a mixture of RIPA buffer (Beyotime, Jiangsu,China) and Protease Inhibitor Cocktail (Roche Applied Science, USA). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to PVDF membranes (Millipore, Billerica, MA). Then the PVDF membranes were blocked with 5% non-fat milk for 1.5 h. The PVDF membranes were incubated with primary antibodies overnight and incubated with a secondary antibody for 1 h after being washed three times the next day. An ECL detection system (Thermo Fisher Scientific) was employed to develop the chemiluminescent signal and the signal were recorded by using ChampChemi500 system (Sagecreation,Beijing, China). All primary antibodies used in this article were listed in Supplementary Table S2. siRNA, shRNA, and gene transfection The siRNAs targeted to TP63, YAP, TEAD1, and FBXO32 were purchased from GenePharma (Shanghai, China). The siRNAs were transfected into SCC cells using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For stable silencing, SCC cells were infected by shRNAs expressing lentivirus and then selected by puromycin. For stable overexpression, SCC cells were infected with Myc-expressing lentivirus and subsequently selected by puromycin. Sequences of siRNAs or shRNAs used in this study were listed in Supplementary Table S3. CRISPR/Cas9-mediated enhancer repression CRISPR interference based on dCas9-KRAB was performed as described previously( 36 , 37 ). First, lenti-KRAB–dCas9-blast was stably introduced into HK1 cells by selection with 6 µg/mL blasticidin (HK1-dCas9-KRAB). The sgRNAs targeted to TP63 binding sites on super enhancers of ITGA6 were designed and inserted into the Lentiguide-puro vector. Then, HK1-dCas9-KRAB cells were infected with lentiGuide-sgRNAs and selected with 2 µg/mL puromycin. The sequences of sgRNA used in this study were listed in Supplementary Table S3. Tumor tissue samples Ten pairs of oral squamous carcinoma samples were obtained from the Department of oral and maxillofacial surgery, The Second Xiangya Hospital, Central South University (Changsha, Hunan, China). The project was approved by the Institute Research Ethics Committee for the use of clinical samples, and each patient signed a consent form to participate in this project. Xenograft tumor formation assays Xenograft tumor formation assays were performed as described previously( 38 ). Nude mice were divided into two major groups according to whether the injected cells were HK1/Ctrl or HK1/shITGA6#2, and the cells in each major group were then divided into five groups and injected with 0.1 ml containing 5×10 5 , 2.5×10 5 , 1.0×10 5 , 5×10 4 , and 5×10 3 tumor cells. The single-cell suspensions were injected into the 5-week-old male nude mice, and after the tumors appeared to be different, the mice were executed and photographed. The mice were subsequently stripped of their tumors, and the tumorigenic rate was counted. All experimental animal procedures in our study were performed following protocols approved by the Animal Welfare Committee of Central South University. Statistical analysis SPSS statistical software was used to analyze the data. Student’s t-test was used for the measurement data of both groups. Two-way ANOVA was used to analyze the viability of the cells. All data were considered statistically significant at P < 0.05. Results BRD4 inhibitors JQ1 and I-BET151 inhibited the stemness features of SCC BRD4, a member of the Bromodomain and Extraterminal (BET) protein family, is widely recognized in cancer for its role in SEs organization and oncogenes expression regulation( 39 ). Inhibition of BRD4 shortcuts the communication between SEs and target promoters( 40 ). Moreover, JQ1 and I-BET151 are effective targeting inhibitors of BET protein (Figure. 1A). To assess the effect of JQ1 and I-BET151, we initially treated diverse cancer cell lines with different concentrations of JQ1 or I-BET151. Tumor sphere formation assays showed that JQ1 and I-BET151 weaken the sphere-forming ability of HK1 and BxPC3 cells (Figure. 1B). RT-qPCR indicated that the expression levels of stem cell markers such as CD44, CD133, and Snail were decreased after treatment with JQ1 and I-BET151(Figure. 1C). CCK8 assay showed that JQ1 and I-BET151 dramatically suppressed cell viability of HK1 and BxPC3 cell lines in a dose- and time-dependent manner (Figure. 1D). Colony formation assays revealed that JQ1 and I-BET151 remarkably reduced colony numbers of HK1 and BxPC3 cells in a dose-dependent manner (Figure. 1E). Therefore, the changes in cell behavior suggested that JQ1 and I-BET151 could inhibit cell stemness. Identification of SEs driven genes associated with stemness features in SCC To address the possible downstream mechanisms underlying JQ1 and I-BET151 inhibiting the stemness features of SCC, we employed RNA-seq to study JQ1 and I-BET151-induced transcriptomic alterations in HK1 cells. RNA-seq indicates that JQ1 and I-BET151 could jointly upregulate and down-regulate some genes (Figure S1 A). According to statistical analysis, we found that JQ1 and I-BET151 jointly down-regulated the expression of 1838 genes and jointly upregulated the expression of 909 genes (Figure. S1B). Combined with SEs landscape of HK1 cells performed in our previous work( 41 ), we identified 91 genes were down-regulated by BRD4 inhibitor treatment and associated with SEs in HK1 cells, suggesting these genes are bona fide SE-driven genes in SCC cells (Figure. S1C and Figure. S1D). Among these 91 SEs-driven genes, PDLIM1, ITGA6, PKPI, FAT2, and CAV2 genes, which are closely related to the occurrence and development of SCC, were selected for representative demonstration. As shown in Figure. S1E, ChIP-seq data analysis revealed that there were super enhancers marked by intensive and broad H3K27ac modifications at nearby the locus of PDLIM1, ITGA6, PKPI, FAT2, and CAV2 genes in HK1 and BxPC3 cells. We further analysis the expression levels of these genes in human SCC samples by analyzing the HNSC-TCGA database. The results showed that these genes' expression was significantly higher in SCC than in normal tissue (Figure. S1F), indicating that acquisition of SEs maybe the cause of abnormal overexpression of oncogenes in SCC. JQ1/I-BET151 regulated the expression of ITGA6 together with SEs To further evaluate the role of SEs in driving oncogene expression in SCC cells, we chose integrin α6 (ITGA6), also known as CD49f, as a representative of SEs-driven oncogene because it is a member of the integrin family and is associated with tumor growth, migration, and stemness( 20 , 21 ). To assess the effect of the BRD4 inhibitor on ITGA6 expression in SCC, we treated HK1 and BxPC3 cells with different concentrations of JQ1 and I-BET.151. RT-qPCR and Western blot showed JQ1 and I-BET151 suppressed the mRNA and protein expression of ITGA6 in a dose-dependent manner (Figure. 2A and Figure. 2B). Using IGV software to analyze H3K27ac ChIP-seq data from HK1 and BxPC3 cells( 25 , 41 ), we found there were four loci marked with high level of H3K27ac at SEs of ITGA6 (Figure. 2C). Then we employed CRISPR/Cas9-mediated enhancer repression to assess the crucial roles of SE in driving ITGA6 transcription. According to sequences of the four H3K27ac enrichment sites, we designed four sgRNAs and introduced these sgRNAs into dCas9-KRAB expressing HK1 cells. RT-qPCR and Western blot assays showed that when sgRNA guided transcription inhibitor KRAB to the H3K27ac enrichment site upstream of ITGA6, the transcription, and expression of ITGA6 was also inhibited (Figure. 2D and Figure. 2E), indicating that the expression of ITGA6 in SCC was regulated by SEs. ITGA6-SE had TP63 dependence Our previous study indicated that ΔNp63α, a prominent isoform of TP63( 2 ), is the core factor enriched in SEs of HK1 cells( 41 ). Other studies also highlighted an essential role of ΔNp63α in shaping the SEs landscape in pancreatic squamous cell carcinoma( 25 , 42 ), prompting us to consider whether SEs of ITGA6 were regulated by ΔNp63α. By analyzing the TCGA database of head and neck SCC, we found that ITGA6 and TP63 expression levels were positively correlated (Figure. 3A). RT-qPCR and Western blot assays showed that ITGA6 and TP63 were expressed in a variety of head and neck tumor cells. Furthermore, we found that ITGA6 was highly expressed in the cells with high ΔNp63α expression and ITGA6 was low expressed in the cells with low ΔNp63α expression (Figure. 3B and Figure. 3C). We used specific siRNAs to silence TP63 in HK1 and BxPC3 cells. RT-qPCR and Western blot assays demonstrated that transient transfection with TP63 siRNAs efficiently suppressed the mRNA and protein levels ITGA6 (Figure. 3D and 3E). Thus, our data suggested that there is a regulatory relationship between TP63 and ITGA6. Using IGV software to analyze TP63 and H3K27ac ChIP-seq data in BxPC3 cells, we found that TP63 protein was highly enriched at the ITGA6-SEs with H3K27ac modifications, whereas the levels of H3K27ac at ITGA6-SEs were dramatically decreased after TP63 knockout by CRISPR/Cas9 approach (Figure. 3F). To further confirm that the ITGA6-SE relies on TP63, we designed four primers according to the binding site. We used the histone acetyl-modified antibody anti-H3K27ac to perform ChIP-qPCR assays on TP63-deficiency cells, revealing that the levels of H3K27ac at SEs of ITGA6 were significantly decreased after TP63 knockdown (Figure. 3G). These results collectively support the notion that the overexpression of ITGA6 in SCC is driven by the SE, which is TP63-dependent. ITGA6 promoted the malignant progression of SCC cells We further asked whether overexpression of ITGA6 contribute to acquisition of stemness features in SCC. ITGA6 expression was analyzed using GEPIA data to determine the role of ITGA6 in SCC development. ITGA6 was highly expressed in various squamous cell carcinomas (Figure. S2A). We analyzed the UALCAN database and found that ITGA6 expression was higher in head and neck SCC and pancreatic SCC samples than in normal control samples (Figure. S2B). Using KM-plot software to analyze the relationship between ITGA6 expression level and prognosis of patients with SCC, we found that patients with higher expression of ITGA6 had decreased overall survival (OS) (Figure. S2C), which indicated that the expression of ITGA6 was closely associated with prognosis of SCC. Moreover, the UALCAN database revealed that ITGA6 was associated with the grade of head and neck SCC and pancreatic SCC. The expression of ITGA6 increased with the deterioration of the tumor (Figure. S2D), but when the patient was in G4 grade, the expression of ITGA6 decreased significantly, while the mechanism was unclear. We examined the expression of ITGA6 in oral squamous carcinoma tissue samples, and the results of RT-qPCR and Western Blot showed that the expression of ITGA6 was higher in oral squamous carcinoma than in matched adjacent non-tumor tissues (Figure. 4A and Figure. 4B).To determine whether overexpression of ITGA6 contribute to acquisition of stemness features in SCC, we exploited shRNAs expressing lentivirus to silence ITGA6 expression. RT-qPCR and Western blot assays showed that two shRNAs targeted to ITGA6 effectively reduced its mRNA and protein levels in HK1 and BxPC3 cells (Figure. 4C and Figure. 4D). Tumor sphere formation assays showed that loss of ITGA6 weakened the sphere-forming ability of HK1 and BXPC3 cells (Figure. 4E), and RT-qPCR assays indicated that ITGA6-knockdown induced downregulation of CD133, CD44 and Snail expression in HK1, BxPC3 cells (Figure. 4F). Drug resistance is a feature of CSCs( 43 ). We treated ITGA6 deficient HK1 and BxPC3 cells with cisplatin at different concentrations for chemotherapy sensitivity analysis. CCK8 assays revealed that inhibition of ITGA6 resulted in decreased cell viability (Figure. S3A). In addition, CCK8 assay showed that ITGA6 deficiency suppressed cell viability in HK1 and BxPC3 cells (Figure. S3B). The scratch wound-healing assays revealed that ITGA6 deficient SCC cells exhibited significantly reduced mobility compared with vector control cells (Figure. S3C). We also performed the Matrigel-coated Boyden chamber invasion assays, and the results demonstrated that ITGA6 deficiency suppressed SCC cell invasiveness in vitro (Figure. S3D). The expression of Ki67 (cell proliferation antigen) in cells was detected using immunofluorescence, which showed that the intensity of Ki67-positive cells decreased after stable interference with ITGA6, demonstrating that ITGA6 can promote the growth of SCC cells (Figure. S3E). Colony formation assays revealed that the loss of ITGA6 also reduced the colony numbers of HK1 and BxPC3 cells (Figure. S3F). We then asked whether ITGA6 promote SCC malignant behavior in vivo using a subcutaneous xenograft tumor model. Nude mice were injected with 0.1 ml containing 5×10 5 , 2.5×10 5 , 1.0×10 5 , 5×10 4 , and 5×10 3 tumor cells, respectively. Our data revealed that when the number of inoculated cells was ≥ 2.5×10 5 /0.1 mL, the tumorigenic rate of HK1 cells was 5/5, while when the number of inoculated cells was ≤ 1.0×10 5 /0.1mL, the tumorigenic rate of ITGA6 deficient HK1 cells was lower than that of the control group, indicating that stable ITGA6 knockdown would lead to a decrease in the number of tumorigenic cells (Figure. 4G). At the endpoint of the experiment, we found xenografts from ITGA6 knockdown cells were much smaller than the vector control cells (Figure. 4H and Figure. 4I). Thus, these data collectively indicated that ITGA6 is closely related to stemness of SCC cells. ITGA6 regulated the stemness of SCC cells through c-Myc active pathway To address the downstream effectors of ITGA6 in regulating stemness of SCC, we employed RNA-seq analysis to screen transcriptomic alteration induced by loss of ITGA6. GSEA analysis revealed that the transcriptome in ITGA6 high HK1 cells was positively correlated with c-Myc activity (Figure. 5A). c-Myc is a classical oncogene and a master regulator of cancer stem cells( 44 ). By analyzing HNSCC-TCGA dataset, we found the expression of c-Myc was higher in SCC tissues than in normal control tissues and was associated with poor prognosis in tumor patients (Figure. S4A and Figure. S4B). Two c-Myc targeting shRNAs expressing lentivirus or an c-Myc cDNA-encoding lentivirus were introduced into HK1 and BxPC3 cells. Western blots assays revealed that two c-Myc targeting shRNAs successfully reduced c-Myc mRNA and protein levels, whereas c-Myc cDNA-encoding lentivirus upregulated c-Myc mRNA and protein levels in HK1 and BxPC3 cells (Figure. S4C). CCK8 assays showed that silencing c-Myc in HK1 and BxPC3 cells suppressed cell growth in vitro. In contrast, forced expression of c-Myc accelerated cell proliferation in SCC cells (Figure. S4D). RT-qPCR assays indicated that stable depletion of c-Myc led to decreased expression of cancer stem cell marker genes, including CD133, Snail. Correspondingly, stable depletion of c-Myc weakened the sphere-forming ability of HK1 and BxPC3 cells (Figure. S4E and Figure. S4F). RT-qPCR assays showed that the mRNA levels of c-Myc in HK1 and BxPC cells were not affected by silencing ITGA6 (Figure. 5B). However, Western blot assays demonstrated that the protein levels of c-Myc were down-regulated after stable deficiency with ITGA6 in HK1 and BxPC3 cells (Figure. 5C). To verify that ITGA6 could promote the malignant progression of SCC by regulating c-Myc, we conducted functional rescue assays. We overexpressed c-Myc in ITGA6-deficient HK1 and BxPC3 cells using an c-Myc-expressing lentivirus. RT-qPCR and Western blot assays revealed that c-Myc -expressing lentivirus upregulated c-Myc mRNA and protein levels in HK1 and BxPC3 cells (Figure. 5D and Figure. 5E). CCK8 assays demonstrated that forced expression of c-Myc rescued cell proliferation in ITGA6 knockdown cells (Figure. 5F). RT-qPCR assays revealed that Snail expression was restored after overexpression of c-Myc in ITGA6 knockdown cells (Figure. 5G). Correspondingly, the sphere-forming ability of HK1 and BxPC3 cells was rescued by overexpression of c-Myc (Figure. 5H), suggesting that ITGA6 promotes stemness features of SCC cells through upregulating c-Myc protein. ITGA6 regulated c-Myc protein through the ubiquitin-proteasome pathway c-Myc, as a classical oncogene, is most notably characterized by its short life span, instability, and susceptibility to degradation, with the ubiquitin-proteasome pathway being the predominant mechanism for c-Myc degradation( 45 ). We analyzed the RNA-seq database and found that ITGA6 deficiency could upregulate FBXO32, which was also revealed by RT-qPCR and Western blot assays (Figure. 6A and Figure. 6B). FBXO32 is an F-box protein that combines SKP1, CUL1, and ROC-1 to form SCF-type E3 ubiquitin ligase( 46 ). And what’s more, it has been reported that FBXO32 could target and degrade c-Myc protein through the proteasome pathway( 47 , 48 ). HNSCC-TCGA data revealed that the expression of FBXO32 was lower in SCC tissues than in normal control tissues (Figure. 6C). We used specific siRNAs to silence FBXO32 in HK1 and BxPC3 cells. RT-qPCR and Western blot assays indicated that FBXO32-knockdown did not affect the mRNA levels of c-Myc but led to the upregulation of c-Myc protein levels. (Figure. 6D and Figure. 6E). Moreover, RT-qPCR and Western blot assays demonstrated that FBXO32 deficiency significantly rescued the protein levels of c-Myc in ITGA6 knockdown cells, with a small effect on its mRNA levels. (Figure. 6F and Figure. 6G). Correspondingly, tumor sphere formation assays showed that FBXO32 deficiency rescued the sphere-forming ability of ITGA6 knockdown cells (Figure. 6H). ITGA6 regulated FBXO32 expression by regulating YAP1 entry into the nucleus To address the mechanism by which ITGA6 regulated FBXO32, we performed further analysis of the RNA-seq database. GSEA analysis revealed that ITGA6 deficiency was positively correlated with YAP conserved signature (Figure. 7A). Yes-associated protein 1(YAP1) is the main effector of the Hippo pathway, which can cooperate with its transcriptional coactivator TAZ to promote cell proliferation, stem cell maintenance and tissue homeostasis ( 49 ). Furthermore, YAP is related to SCC cells' deformation and metastasis potential ( 50 ). Considering that the Hippo-YAP/TAZ pathway can be regulated by the cytoskeleton and is the main receptor of cells to mechanical forces( 51 , 52 ), we observed the changes in the mechanical characteristics of ITGA6 deficient cells. Using atomic force microscopy, we found that the cell surface roughness (Rq value) decreased after ITGA6 knockdown in HK1 and BxPC3 cells, indicating that the cell surface became smoother, resulting in reduced tumor motility (Fig. 7 B). In addition, after stably knockdown ITGA6, the cell stiffness value increased (Fig. 7 C), that is, the cell hardness increased, indicating that the cell deformation ability decreased, further proving that knockdown of ITGA6 can inhibit the migration and invasion of SCC cells. RT-qPCR and Western blot experiments showed that the knockdown of ITGA6 did not affect YAP1's mRNA and protein levels (Figure. 7D and Figure. 7E). Immunofluorescence assays showed that YAP1 protein was localized in the cytoplasm where it was colocalized with ITGA6 (Figure. S5A). Cytoplasmic and nucleus separation assays showed that ITGA6 deficiency could promote YAP1 entry into the nucleus (Figure. 7F). Western blot assays revealed that two siRNAs targeted to YAP1 effectively reduced their protein levels in HK1 and BxPC3 cells (Fig. 7 G). We found that YAP1 co-knockdown could reverse the upregulation of FBXO32 expression induced by ITGA6 knockdown (Figure. 7H and Figure. 7I), suggesting induction of FBXO32 by loss of ITGA6 is mediated by YAP1. Furthermore, tumor sphere formation assays showed that co-knockdown of YAP1 could rescue the sphere-forming ability of ITGA6-knockdown cells (Figure. S5B). Correspondingly, RT-qPCR assays indicated that co-knockdown of YAP1 could rescue CD133, CD44, and Snail expression in ITGA6 deficient cells (Figure. S5C). CCK8 assays showed that co-knockdown of YAP1 in ITGA6 deficient cells could reverse the inhibition of cell viability (Figure. S5D). These data suggested that ITGA6 may enhance squamous carcinoma's stemness and malignant behavior by binding to YAP1 and inhibiting YAP1 entry into the nucleus. The transcriptional regulator YAP1 does not have a DNA-binding domain, and it uses the TEAD1 (TEA domain) family of transcription factors predominantly to elicit most of their biologically relevant gene expression programs( 53 ). We used specific siRNAs to silence TEAD1 in HK1 and BxPC3 cells. RT-qPCR assays demonstrated that transfection with siRNAs efficiently suppressed the mRNA level of TEAD1, and the expression level of FBXO32 was downregulated after the TEAD1 knockdown in HK1, BxPC3 cells (Fig. 7 J). ChIP-qPCR assays showed that TEAD1 binds to the promoter region of FBXO32(Figure.7K). Thus, these data suggested that ITGA6 could affect the malignant behavior of SCC cells through YAP1/TEAD1, FBXO32, and c-Myc axes. Discussion In this study, we uncovered that overexpression of ITGA6 driven by TP63-dependent super enhancer enhances stemness features in SCC through regulating a YAP1-FBXO32-c-Myc axis, whereas disruption of super enhancer driven ITGA6 by BRD4 inhibitors suppresses stemness features in SCC (Fig. 8 ). Our study suggested targeting super enhancer may provide a therapeutic strategy for eliminating cancer stem cells. Enhancers, a DNA sequence that can bind to transcription factors and enhance the transcription of target genes, play an important role in cell differentiation, body development, and tumorigenesis( 54 ). Activated enhancers are usually enriched for histone H3K4me1/2 and H3K27ac modifications ( 55 ). In 2013, American scientists proposed the concept of SEs ( 56 ). Compared with ordinary enhancers, SEs can promote gene transcription more effectively( 11 ). Scientists have found that many key cancer genes' expressions are regulated by SEs. For example, in melanoma, researchers have found that SEs drive the high expression of AMIGO2, which promotes the malignant progression of tumors( 57 ). Disruption of SEs often brings many changes to the cell. We found that JQ1 and I-BET151 treatment could reduce the stemness and inhibit the malignant behavior of SCC cells. These results suggest that cancer stemness genes may be regulated by SEs and the disruption of SEs is a promising approach to eliminate CSCs in SCC. This hypothesis is also supported by other’s observation that cancer stemness genes are regulated by SEs in SCC ( 16 ) We identified 91 SEs driven genes in HK1 cells. According to the TCGA data, most of these SEs driven-genes are overexpressed in HNSCC samples and are associated with poor prognosis, highlighting super-enhancer-associated genes contribute to cancer progression( 14 , 58 ). Among them, ITGA6, as a member of integrin, participates in regulating various life activities, and its abnormal expression is related to the occurrence and development of tumors. Many studies have shown that ITGA6 is closely related to the malignant progression of tumors ( 59 , 60 ). In addition, ITGA6 expression has been detected in more than 30 stem cells, and ITGA6 has been identified as the only gene co-expressed in embryonic stem cells, embryonic neural stem cells, and hematopoietic stem cells( 61 , 62 ). These studies suggested that ITGA6 plays a very important role in stem cell biology. We provide strong evidence showing that ITGA6 is overexpressed in HNSCC samples and associated with unfavorable prognosis of HNSCC patients. Notably, CRISPR/Cas9-mediated enhancer repression confirmed that the expression of ITGA6 in SCC cells is tightly regulated by SEs. We also provide experimental evidence confirming that high expression of ITGA6 is required for maintenance of tumor initiating cells in SCC. Thus, ITGA6 is a bona fide SE-driven cancer stemness gene. Our and other’s studies established an essential role of ΔNp63α in shaping SCC specific SEs landscape( 2 , 25 , 41 , 42 ). In this study, we found that SEs at ITGA6 locus relies on ΔNp63α, because depletion of ΔNp63α by CRISPR/Cas9 or RNAi resulted in loss of H3K27ac modification at SEs of ITGA6 and consequent reduction of ITGA6 expression level. As the prominent isoform of TP63 in SCC, ΔNp63α is a well-recognized lineage survival oncogene ( 2 , 41 ). Our study indicates that ΔNp63α may regulate cancer stemness genes by shaping SEs, which is in consistent with other’s observation that ΔNp63α contributes to stemness characteristics in SCC( 16 ). RNA-Seq analysis suggested that ITGA6 high HK1 cells exhibit high c-Myc activity. c-Myc is a nuclear transcription factor which has a pivotal role in maintaining cancer stem cell populations( 63 , 64 ). One of the most prominent features of c-Myc is its short life span, instability, and easy degradation. Ubiquitin- proteasome pathway is the main mechanism of c-Myc degradation( 45 ). In our study, we found that ITGA6 deficiency did not regulate the mRNA levels of c-Myc but caused a decrease in c-Myc protein levels. Overexpression of c-Myc in ITGA6 knockdown cells can restore the tumor sphere forming ability and the expression of CSCs markers in SCC cells. Accelerated c-Myc protein degradation in ITGA6 deficient cells is due to upregulation of FBXO32, a known E3 ligase targeting c-Myc for ubiquitin-proteasomal degradation( 47 ). No evidence suggest ITGA6 could directly mediate gene transcription. Thus, upregulation of the mRNA level of FBXO32 upon inhibition of ITGA6 should be a consequence of altered signal transduction in ITGA6 deficient cells. GSEA analysis on transcriptomic alterations induced by loss of ITGA6 suggested YAP signaling involved. We found inhibition of ITGA6 led to increased YAP1 entry into the nucleus. We further provide evidence showing that FBXO32 is a direct target gene of YAP1/TEAD1 complex. Functionally, silencing YAP1 in ITGA6-deficient cells resulted in reduction of the level of FBXO32, but restoration of c-Myc protein and stemness features of SCC cells, indicating that ITGA6 sustains cancer stemness through preventing activation of YAP1/TEAD1-FBXO32 signaling. Many previous studies have shown that hyper-activation of YAP signaling pathway promotes the expansion of cancer stem cells and drug resistance of cancer cells( 65 , 66 ). However, increasing evidence suggests that YAP1 plays a dual role in tumorigenesis and progression. Some scientists have found that cancers can be divided into YAP-on and YAP-off categories according to the presence or absence of YAP protein. For YAP-on cancers, cancer cells need YAP to grow and survive, while for YAP-off cancers, the presence of YAP can inhibit the growth of cancer cells ( 67 ). In esophageal squamous cell carcinoma, YAP1 functions as a tumor suppressor, and patients with a high YAP1 expression have better overall survival( 68 ). There are also controversies about roles of YAP1 in self-renewal vs. differentiation. It has been shown that in mouse embryonic stem cells, YAP1 is indispensable for self-renewal but required for differentiation. Nuclear translocation of YAP1 in mouse embryonic stem cells leads to disruption of self-renewal and triggering differentiation( 69 ). In this study, increase of nuclear translocation of YAP1 attenuated stemness features and malignant bechaviors of SCC cells upon loss of ITGA6, suggesting a tumor suppressive role of YAP1 in this context. We believe that whether YAP1 plays oncogenic or tumor suppressive roles in cancers may depend on the specific cellular contexts. To date, we don’t have a clear idea on how ITGA6 in regulating the nuclear translocation and activity of YAP1. It has been shown that phosphorylation of YAP by active LATS1/2 kinases causes sequestration of YAP in cytoplasm via 14-3-3 protein( 70 ). We observed that ITGA6 colocalized with YAP1 protein in cytoplasm of SCC cells. We speculate that ITGA6 may bind with YAP1 and exert a similar function as 14-3-3 protein. Another possibility is that increase of YAP1 nuclear translocation is triggered by alterations of the biomechanical properties of SCC cells upon silencing ITGA6, since the YAP/TAZ sense and respond to biomechanical signals( 71 ). Conclusions In summary, our results demonstrate that BRD4 inhibitors suppresses stemness features in SCC cells. We identified a panel of SEs-driven genes associated with stemness features in SCC. Among these SEs-driven genes, ITGA6 is driven by ΔNp63α-dependent SEs and overexpressed in SCC. Elevated expression of ITGA6 stabilizes c-Myc protein via inhibition on YAP1/TEAD1-FBXO32 signal, which target c-Myc for degradation (Fig. 8 ). Our study provides a new idea for epigenetically targeting CSCs. Abbreviations squamous cell carcinoma (SCC); super enhancers (SEs); cancer stem cells (CSCs); Bromodomain Containing 4 (BRD4); Integrin α6 (ITGA6); phosphate buffered saline (PBS); Cell Counting Kit-8 (CCK-8); atomic force microscope (AFM); Yes1 associated transcriptional regulator (YAP1); TEA domain transcription factor 1 (TEAD1); F-Box Protein 32 (FBXO32); Head and neck squamous cell carcinoma (HNSCC); The Cancer Genome Atlas (TCGA); clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). Declarations Ethics approval and consent to participate Approval for the use of clinical samples and information was obtained from each patient and the Research Ethics Committee of Central South University. Written informed consent was received prior to patient participation. The animal experiments were conducted according to the protocol approved by the Animal Welfare Committee of Central South University. Consent for publication All authors have read the manuscript and agree to publish. Availability of Data and Materials The datasets and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors have declared that no conflict of interest exists. Funding This study was supported in part by grants from The National Natural Science Foundation of China (82272631, 82072596, 82173339, 82172766, 81872278), the National “111” Project (Project #111-2-12), the Hunan Provincial Key Research and Development Program (2022SK2026), the Natural Science Foundation of Hunan Province, China (2020JJ4920, 2020JJ4838, 2020JJ4766, 2020JJ3055), the Scientific Research Project of Hunan Provincial Health Commission (20201067, 20201040), the Beijing Xisike Clinical Oncology Research Foundation (Y-HR2020ZD-0052), the Open Funds of State Key Laboratory of Oncology in south China (HN2021–07). 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Yi","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mei","middleName":"","lastName":"Yi","suffix":""}],"badges":[],"createdAt":"2023-06-03 10:50:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3017766/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3017766/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":39863923,"identity":"7a0c462d-6cd3-4a21-911b-5cf436ae7e4d","added_by":"auto","created_at":"2023-07-11 14:53:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":854641,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBRD4 inhibitors JQ1 and I-BET151 inhibited the stemness features of SCC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eChemical structure of JQ1 and I-BET151. \u003cstrong\u003eB.\u003c/strong\u003e Tumor sphere formation assays. Cells were cultured using a sphere-forming medium containing different concentrations of JQ1 or I-BET151, and larger microspheres were visible after about 1 week, photographed, and counted. \u003cstrong\u003eC.\u003c/strong\u003e The mRNA levels of CD133, CD44, and Snail were detected by qPCR. \u003cstrong\u003eD.\u003c/strong\u003e Cell viability assays. HK1 and BXPC3 cells were exposed to different concentrations of JQ1 or I-BET151.\u003cstrong\u003e E. \u003c/strong\u003eColony formation assays. HK1 and BXPC3 Cells were treated with different doses of JQ1 or I-BET151 for 24 h, followed by incubation in a fresh culture medium for about two weeks. Colonies were visualized after crystal purple staining. Data values are expressed as mean ± SD.\u003cstrong\u003e \u003c/strong\u003e*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3017766/v1/849c626c55e12c54026f0b2a.png"},{"id":39862560,"identity":"e38dcb19-4870-41b1-825c-fb7af68f73e0","added_by":"auto","created_at":"2023-07-11 14:45:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":614460,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJQ1/IBET151 and SEs regulated the expression of ITGA6 in SCC cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eThe mRNA levels of ITGA6 in cells treated with various doses of JQ1/I-BET151 were measured by RT-qPCR assays. \u003cstrong\u003eB.\u003c/strong\u003e The protein levels of ITGA6 in cells treated with various doses of JQ1/I-BET151 were measured by Western blot assays. \u003cstrong\u003eC.\u003c/strong\u003e IGV software analysis of ITGA6 upstream SEs enrichment sites. \u003cstrong\u003eD.\u003c/strong\u003e The mRNA levels of ITGA6 were measured by RT-qPCR assays. \u003cstrong\u003eE. \u003c/strong\u003eThe protein levels of ITGA6 were measured by Western blot assays. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3017766/v1/41b4df7c819fd3757020164d.png"},{"id":39862559,"identity":"0a487d05-d3ac-440f-b878-f46c295726b6","added_by":"auto","created_at":"2023-07-11 14:45:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":375251,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITGA6-SE had TP63 dependence.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eCorrelation analysis of ITGA6 with TP63 in HNSCC. \u003cstrong\u003eB.\u003c/strong\u003e The mRNA levels of TP63 and ITGA6 were determined by RT-qPCR assays. \u003cstrong\u003eC.\u003c/strong\u003e The protein levels of TP63 and ITGA6 were determined by Western blot assays. \u003cstrong\u003eD. \u003c/strong\u003eThe mRNA levels of TP63 and ITGA6 after siRNA silencing TP63 in HK1 and BXPC3 cells were measured by RT-qPCR assays. \u003cstrong\u003eE. \u003c/strong\u003eThe protein levels of TP63 and ITGA6 after siRNA silencing TP63 in HK1 and BXPC3 cells were measured by Western blot assays. \u003cstrong\u003eF.\u003c/strong\u003eIGV software analysis of TP63 in combination with ITGA6-SE. \u003cstrong\u003eG.\u003c/strong\u003e ITGA6-SE H3K27ac activity after stable interference with TP63 was measured by ChIP-qPCR assays. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3017766/v1/ad79b0d1f0086eb8f92e5c5e.png"},{"id":39864841,"identity":"89f52ea4-8062-4994-a7bf-2837c7bd4948","added_by":"auto","created_at":"2023-07-11 15:01:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":931361,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITGA6 regulated tumor stemness in SCC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e RT-qPCR of ITGA6 in ten matched pairs of adjacent non-tumor (N) and tumor (T) tissues (n = 10).\u003cstrong\u003e B.\u003c/strong\u003eWestern Bloting of ITGA6 in ten matched pairs of adjacent non-tumor (N) and tumor (T) tissues (n = 10).\u003cstrong\u003e C.\u003c/strong\u003e The mRNA levels of ITGA6 in HK1 or BxPC3 cells were measured by RT-qPCR assays. \u003cstrong\u003eD.\u003c/strong\u003e The protein levels of ITGA6 in HK1 or BXPC3 cells were detected by Western blot assays. \u003cstrong\u003eE.\u003c/strong\u003e Tumor sphere formation assays. \u003cstrong\u003eF.\u003c/strong\u003e \u0026nbsp;The mRNA levels of ITGA6, CD133, CD44, and Snail were measured by RT-qPCR assays. \u003cstrong\u003eG.\u003c/strong\u003e The tumor formation rates of different groups were counted after xenograft assays. \u003cstrong\u003eH.\u003c/strong\u003e The picture of xenograft tumors of nude mice.\u003cstrong\u003e I.\u003c/strong\u003e Tumor volume in different groups of nude mice. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3017766/v1/04863e85797e35810fde1e94.png"},{"id":39862565,"identity":"eba3d21c-18ec-467a-8f07-bd6f4ee95eaf","added_by":"auto","created_at":"2023-07-11 14:45:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":669023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITGA6 promoted the malignant progression of SCC by regulating c-Myc.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eGSEA analysis indicated that ITGA6 could regulate the activity of c-Myc. A GSEA analysis revealed that ITGA6-knockdown-induced transcriptomic alterations were correlated with c-Myc active pathway. \u003cstrong\u003eB.\u003c/strong\u003e RT-qPCR demonstrated the expression level of c-Myc after ITGA6 knockdown. \u003cstrong\u003eC. \u003c/strong\u003eThe protein levels of c-Myc were measured by Western blot. \u003cstrong\u003eD.\u003c/strong\u003e The mRNA levels of c-Myc in c-Myc expressing lentivirus infected cells were measured by RT-qPCR. \u003cstrong\u003eE.\u003c/strong\u003e The protein levels of c-Myc were measured by Western blot. \u003cstrong\u003eF. \u003c/strong\u003eCell viability assays show that overexpression of c-Myc could rescue the reduced cell activity caused by ITGA6 knockdown. \u003cstrong\u003eG. \u003c/strong\u003eThe mRNA levels of c-Myc and Snail were measured by RT-qPCR. \u003cstrong\u003eH.\u003c/strong\u003e Tumor sphere formation assays demonstrated that overexpression of c-Myc could rescue the reduced cell sphere-forming ability caused by ITGA6 knockdown. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3017766/v1/bf5db004e9d9835d86e58f01.png"},{"id":39862562,"identity":"6fbc6a6d-be1e-4442-868c-1e80d6d71d5f","added_by":"auto","created_at":"2023-07-11 14:45:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":579940,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITGA6 regulated c-Myc through FBXO32.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eRT-qPCR revealed that ITGA6 deficiency could upregulate the mRNA levels of FBXO32. \u003cstrong\u003eB.\u003c/strong\u003e The protein levels of FBXO32 were measured by Western blot. \u003cstrong\u003eC. \u003c/strong\u003eThe mRNA levels of FBXO32 were assessed in the HNSCC-TCGA database. \u003cstrong\u003eD. \u003c/strong\u003eThe mRNA levels of FBXO32 and c-Myc were measured by RT-qPCR. \u003cstrong\u003eE. \u003c/strong\u003eThe protein levels of c-Myc were measured by Western blot.\u003cstrong\u003e F. \u003c/strong\u003eRT-qPCR assays showed that FBXO32 knockdown rescued MYC expression in ITGA6 deficiency cells. \u003cstrong\u003eG. \u003c/strong\u003eThe protein levels of c-Myc were measured by Western blot.\u003cstrong\u003e H.\u003c/strong\u003e Tumor sphere formation assays demonstrated that FBXO32-knockdown could rescue the cell sphere-forming ability in ITGA6 deficiency cells. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3017766/v1/e5a832e2e7e55d03a35e8acd.png"},{"id":39862563,"identity":"d88d4578-2e7d-4c62-b679-306381b71906","added_by":"auto","created_at":"2023-07-11 14:45:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":752383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITGA6 regulated FBXO32 expression by regulating YAP1 entry into the nucleus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eGSEA analysis indicated that ITGA6 could regulate YAP conserved signature.\u003cstrong\u003e B.\u003c/strong\u003eCell surface roughness changes after ITGA6-knockdown are shown by atomic force microscopy. Top, Cell surface photography; bottom, Statistical Chart. \u003cstrong\u003eC.\u003c/strong\u003eCell surface hardness changes after ITGA6-knockdown is shown by atomic force microscopy.\u003cstrong\u003e \u003c/strong\u003eTop, Cell surface photography; bottom, Statistical Chart. \u003cstrong\u003eD.\u003c/strong\u003eThe mRNA levels of ITGA6 and YAP1 were measured by RT-qPCR assays.\u003cstrong\u003e E. \u003c/strong\u003eThe protein levels of YAP1 and TAZ were measured by Western Blot assays. \u003cstrong\u003eF. \u003c/strong\u003eThe protein levels of YAP1, TAZ, and Histone3 were measured by Western blot assays.\u003cstrong\u003eG.\u003c/strong\u003e The protein levels of YAP1 in ITGA6/YAP1 deficiency cells were determined by Western blot assays. \u003cstrong\u003eH. \u003c/strong\u003eThe mRNA levels of YAP1 and FBXO32 in ITGA6/YAP deficiency cells were measured by RT-qPCR assays. \u003cstrong\u003eI. \u003c/strong\u003eThe protein levels of YAP1 and FBXO32 were measured by Western blot assays. \u003cstrong\u003eJ.\u003c/strong\u003e The mRNA levels of TEAD1 and FBXO32 were measured by RT-qPCR assays. \u003cstrong\u003eK.\u003c/strong\u003eThe binding of TEAD1 to the FBXO32 promoter was detected by ChIP-qPCR. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3017766/v1/5d45b5b806ea1d04c954fb3f.png"},{"id":39863925,"identity":"fd998e7a-0005-4ee7-bbd9-8feeb98e1410","added_by":"auto","created_at":"2023-07-11 14:53:14","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":235361,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of the mechanism by which SEs-driven ITGA6 enhances the malignant behavior of SCC cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn SCC cells, TP63-dependent SEs could drive high expression of ITGA6. After entering into the cytoplasm, ITGA6 could bind to YAP and lock YAP to the cytoplasm. JQ1 or I-BET151 can inhibit the transcription and expression of ITGA6 by interfering with the SEs, promoting YAP1 entry into the nucleus, and upregulating the expression of FBXO32 via TEAD1. FBXO32, an important component of E3 ubiquitin ligase, targets c-Myc for degradation, ultimately leading to reduced malignant behavior in squamous carcinomas. This figure is drawn by Figdraw.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3017766/v1/e8fa69d280790b198559659a.png"},{"id":42432795,"identity":"d92af57b-2064-4067-a155-d0f0960ef8ea","added_by":"auto","created_at":"2023-08-31 14:40:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5405204,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3017766/v1/89fa166d-6dab-47d7-930e-ebda34a73dc7.pdf"},{"id":39862566,"identity":"d8f0101e-99a2-4f93-a15d-7420823965a4","added_by":"auto","created_at":"2023-07-11 14:45:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3960608,"visible":true,"origin":"","legend":"","description":"","filename":"supportingdata2023.docx","url":"https://assets-eu.researchsquare.com/files/rs-3017766/v1/efc89697823d69fc3ab08b10.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Super-enhancer-driven ITGA6 enhances stemness features of squamous cell carcinoma through stabilizing c-myc protein","fulltext":[{"header":"Background","content":"\u003cp\u003eSquamous cell carcinoma (SCC) is one of the most common solid neoplasms worldwide, mainly arising within the stratified epithelium of skin, lung, esophagus, aerodigestive or genitourinary tracts(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). SCC is an aggressive malignancy, prone to metastasis, highly malignant, and has a poor prognosis, threatening human safety (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). However, the underlying mechanisms driving the malignant progression of SCC are not well understood.\u003c/p\u003e \u003cp\u003eIn the development of tumorigenesis, cancer stem cells (CSCs) are an important factor leading to tumor initiation, drug resistance, recurrence, and metastasis. Eliminating tumor stem cells is an effective treatment for tumors(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). CSCs mainly originate from the accumulation of genetic mutations in primitive cells, activation of self-renewal programs, and inactivation of senescence and apoptosis programs (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In addition, aberrant epigenetic alterations, including DNA methylation and histone modifications, play crucial roles in transforming normal stem cells to CSCs or the acquisition of stem-like features in cancer cells(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Enhancers emerge as a key noncoding cis-regulatory DNA sequence that can affect gene transcription. By acting on promoters, enhancers can regulate promoter activity and activate or enhance gene transcription(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Super enhancers (SEs) are large clusters of transcriptional enhancers that can play a prominent role in driving the expression of cell-type-specific genes through interacting with enriched transcription factors, co-factors, RNA polymerase II, and noncoding RNAs(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In human cancers, acquisition of SEs at oncogenes loci is an alternative mechanism for abnormal activation of oncogenes(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). SEs are highly sensitive to the BRD4 inhibitor JQ1 and easily disturbed, so they can be a target for drug intervention(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). SEs have been shown to affect the transcription of CSCs in SCC, thereby influencing the malignant behavior of the tumor (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Thus, identifying SEs associated genes contributed to maintenance of CSCs characteristics may provide insights into the role of SEs in controlling stemness of CSCs and development of antitumor drugs.\u003c/p\u003e \u003cp\u003eIntegrins are the main cell adhesion receptors of extracellular matrix components, and they are a family of 24 transmembrane heterodimers generated from a combination of 18 α integrin and 8 β integrin subunits(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). As a transmembrane signaling receptor, integrin can mediate epithelial cell adhesion, affect cell growth and differentiation, and is associated with tumor progression and poor prognosis in many epithelial malignant tumors(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Integrin α6 (ITGA6), also known as CD49f, is a member of the integrin family, which has been detected to be abnormally expressed in various tumors such as breast cancer, lung cancer, and liver cancer(\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated that disruption of SEs by BET inhibitors impaired the stemness features and malignant behaviors of SCC cells. We identified highly confident SEs associated genes in SCC cell line by integrative analysis H3K27ac ChIP-seq and RNA-seq following BET inhibitors treatment. Among these SEs associated genes, ITGA6 is highly expressed in various SCCs and associated with poor prognosis. Loss of ITGA6 accelerates proteasomal degradation of c-myc protein, leading to impairment of stemness features of SCCs cells. Inhibition of ITGA6 promotes nuclear translocation of YAP1 and activates the transcription of FBXO32, which acts as an E3 ligase targeting to c-myc protein.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines, cell culture and inhibitors\u003c/h2\u003e \u003cp\u003eHK1 is a highly differentiated squamous carcinoma cell from nasopharynx maintained in our lab(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). BxPC3 is a pancreatic squamous carcinoma cell purchased from the National Infrastructure of Cell Line Resource (Shanghai, China)(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Cells were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, USA) and maintained in a humidified incubator consisting of 5% CO2 and 95% air at 37℃. JQ-1 (HY-13030) and I-BET151 (HY-13235) were purchased from MedChem Express (MCE, Monmouth Junction, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCCK8 assay\u003c/h2\u003e \u003cp\u003eCCK8 assay was performed according to formerly described(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Briefly, cells were seeded into 96-well plates at a density of 1\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/100\u0026micro;L. Cells were allowed to grow for 0, 24, 48, 72, 96, and 120 h and were counted by using Cell Counting Kit (CCK-8). Besides, when cells were treated with drugs, 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells were seeded into 96-well plates supplemented 200\u0026micro;L culture medium containing the indicated dosage of JQ1 or I-BET151, then the cells were allowed to grow for 24 h and were measured by using CCK-8 assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assay\u003c/h2\u003e \u003cp\u003eColony formation assay was performed as described previously(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). HK1 and BxPC3 cells were seeded into 24-well plates at a density of 1 \u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well and cultured for about two weeks. When cells were treated with drugs, the original medium would be changed to a fresh complete medium containing the indicated dosage of JQ1 or I-BET151 after the cell in adherent. Finally, colonies were fixed with 4% paraformaldehyde and visualized with 1% crystal violet. These assays were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTumor sphere formation assay\u003c/h2\u003e \u003cp\u003eTumor sphere formation assay was performed as previously described(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Briefly, 4\u0026times;10\u003csup\u003e3\u003c/sup\u003e HK1 or BxPC3 cells were cultured in ultra-low adhesion six-well plates using sphere-forming medium-DMEM/F12 (Gibco)\u0026thinsp;+\u0026thinsp;EGF(20 ng/ml) (Gibco)\u0026thinsp;+\u0026thinsp;bFGF(10 ng/ml) (PeproTech)\u0026thinsp;+\u0026thinsp;B27(1\u0026times;). Cells were treated with drugs by adding different concentrations of chemicals to the culture medium. After about 7\u0026ndash;10 days, larger microspheres were observed and counted under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell migration and invasion assays\u003c/h2\u003e \u003cp\u003eCell migration and invasion assays were performed as described previously(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells in serum-free medium were seeded into transwell inserts precoated with or without XXXatrigel. Then the inserts were put into the chamber of the 24-well plates which added with 700 \u0026micro;L culture medium containing 15% FBS. Then the transwells were incubated at 37℃ for 24-48h to allow tumor cells migrating or invading across the transwell membrane. The migrated or invaded tumor cells were then fixed with 4% paraformaldehyde and visualized with crystal violet. The number of migrated or invaded tumor cells were counted and photographed under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAtomic Force Microscopy (AFM)\u003c/h2\u003e \u003cp\u003eThe biophysical properties of SCC cells were measured by AFM (JPK NanoWizard 4 BioScience, JPK Instruments, Germany) as described previously(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Briefly, cells were fixed in 2% glutaraldehyde for 45 s, 4% paraformaldehyde for 20 min, and then washed with PBS. AFM scanning were performed by using the probe HYDRA6V-100NG (AppNano, CA, USA) with a spring constant of 0.292 N/m. Cell adhesion, stiffness, and Rq were analyzed with JPK software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and real-time reverse-transcription PCR (RT-qPCR)\u003c/h2\u003e \u003cp\u003eTotal intracellular RNA was extracted by using TRIzol Reagent (Life Technologies) as previously described(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). For reverse transcription, 3 \u0026micro;g of total RNA samples were digested with DNase I (Takara, Beijing, China) to remove the trace amount of genomic DNA, then the RNA samples were reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kits (Thermo Fisher Scientific, Beijing, China). SYBR Green reagent (Bimake, Shanghai, China) was employed to perform Real-Time PCR assay by using CFX96 Touch\u0026trade; Real-Time PCR Detection System (Bio-Rad Laboratories, Richmond, CA, USA). The 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method was used to evaluate the relative expression levels of genes. PCR primers used in qPCR assays are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA-Seq\u003c/h2\u003e \u003cp\u003eRNA samples for RNA-Seq were prepared as described above, and sequencing was performed by an Illumina HiSeq platform (San Diego, CA, USA). The NOISeq method was employed to determine the differentially expressed genes with fold changes\u0026thinsp;\u0026ge;\u0026thinsp;2 (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Gene set enrichment analysis (GSEA) was performed to analyze the result(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChIP-qPCR\u003c/h2\u003e \u003cp\u003eFormaldehyde-cross-linked protein-DNA complexes were prepared from HK1, and BxPC3 live cells and cut into 200\u0026ndash;1000 bp DNA fragments by ultrasound. Protein-DNA complexes were precipitated using anti-H3K27ac (active motif; Cat No. 39685), anti-TEAD1() antibody. The normal human immunoglobulin G (IgG) was used as negative control for antibody. The DNA fragments precipitated with antibodies were purified and determined by qPCR with specific Primers. Primers specific to each segment of interest are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blot analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWestern Blot analysis was performed according to previously described(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Cells were lysed with a mixture of RIPA buffer (Beyotime, Jiangsu,China) and Protease Inhibitor Cocktail (Roche Applied Science, USA). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to PVDF membranes (Millipore, Billerica, MA). Then the PVDF membranes were blocked with 5% non-fat milk for 1.5 h. The PVDF membranes were incubated with primary antibodies overnight and incubated with a secondary antibody for 1 h after being washed three times the next day. An ECL detection system (Thermo Fisher Scientific) was employed to develop the chemiluminescent signal and the signal were recorded by using ChampChemi500 system (Sagecreation,Beijing, China). All primary antibodies used in this article were listed in Supplementary Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003esiRNA, shRNA, and gene transfection\u003c/h2\u003e \u003cp\u003eThe siRNAs targeted to TP63, YAP, TEAD1, and FBXO32 were purchased from GenePharma (Shanghai, China). The siRNAs were transfected into SCC cells using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For stable silencing, SCC cells were infected by shRNAs expressing lentivirus and then selected by puromycin. For stable overexpression, SCC cells were infected with Myc-expressing lentivirus and subsequently selected by puromycin. Sequences of siRNAs or shRNAs used in this study were listed in Supplementary Table S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCRISPR/Cas9-mediated enhancer repression\u003c/h2\u003e \u003cp\u003eCRISPR interference based on dCas9-KRAB was performed as described previously(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). First, lenti-KRAB\u0026ndash;dCas9-blast was stably introduced into HK1 cells by selection with 6 \u0026micro;g/mL blasticidin (HK1-dCas9-KRAB). The sgRNAs targeted to TP63 binding sites on super enhancers of ITGA6 were designed and inserted into the Lentiguide-puro vector. Then, HK1-dCas9-KRAB cells were infected with lentiGuide-sgRNAs and selected with 2 \u0026micro;g/mL puromycin. The sequences of sgRNA used in this study were listed in Supplementary Table S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTumor tissue samples\u003c/h2\u003e \u003cp\u003eTen pairs of oral squamous carcinoma samples were obtained from the Department of oral and maxillofacial surgery, The Second Xiangya Hospital, Central South University (Changsha, Hunan, China). The project was approved by the Institute Research Ethics Committee for the use of clinical samples, and each patient signed a consent form to participate in this project.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eXenograft tumor formation assays\u003c/h2\u003e \u003cp\u003eXenograft tumor formation assays were performed as described previously(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Nude mice were divided into two major groups according to whether the injected cells were HK1/Ctrl or HK1/shITGA6#2, and the cells in each major group were then divided into five groups and injected with 0.1 ml containing 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e, 2.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e, 1.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e, 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e, and 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e tumor cells. The single-cell suspensions were injected into the 5-week-old male nude mice, and after the tumors appeared to be different, the mice were executed and photographed. The mice were subsequently stripped of their tumors, and the tumorigenic rate was counted. All experimental animal procedures in our study were performed following protocols approved by the Animal Welfare Committee of Central South University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSPSS statistical software was used to analyze the data. Student\u0026rsquo;s t-test was used for the measurement data of both groups. Two-way ANOVA was used to analyze the viability of the cells. All data were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBRD4 inhibitors JQ1 and I-BET151 inhibited the stemness features of SCC\u003c/h2\u003e \u003cp\u003eBRD4, a member of the Bromodomain and Extraterminal (BET) protein family, is widely recognized in cancer for its role in SEs organization and oncogenes expression regulation(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Inhibition of BRD4 shortcuts the communication between SEs and target promoters(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Moreover, JQ1 and I-BET151 are effective targeting inhibitors of BET protein (Figure. 1A). To assess the effect of JQ1 and I-BET151, we initially treated diverse cancer cell lines with different concentrations of JQ1 or I-BET151. Tumor sphere formation assays showed that JQ1 and I-BET151 weaken the sphere-forming ability of HK1 and BxPC3 cells (Figure. 1B). RT-qPCR indicated that the expression levels of stem cell markers such as CD44, CD133, and Snail were decreased after treatment with JQ1 and I-BET151(Figure. 1C). CCK8 assay showed that JQ1 and I-BET151 dramatically suppressed cell viability of HK1 and BxPC3 cell lines in a dose- and time-dependent manner (Figure. 1D). Colony formation assays revealed that JQ1 and I-BET151 remarkably reduced colony numbers of HK1 and BxPC3 cells in a dose-dependent manner (Figure. 1E). Therefore, the changes in cell behavior suggested that JQ1 and I-BET151 could inhibit cell stemness.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of SEs driven genes associated with stemness features in SCC\u003c/h2\u003e \u003cp\u003eTo address the possible downstream mechanisms underlying JQ1 and I-BET151 inhibiting the stemness features of SCC, we employed RNA-seq to study JQ1 and I-BET151-induced transcriptomic alterations in HK1 cells. RNA-seq indicates that JQ1 and I-BET151 could jointly upregulate and down-regulate some genes (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). According to statistical analysis, we found that JQ1 and I-BET151 jointly down-regulated the expression of 1838 genes and jointly upregulated the expression of 909 genes (Figure. S1B). Combined with SEs landscape of HK1 cells performed in our previous work(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), we identified 91 genes were down-regulated by BRD4 inhibitor treatment and associated with SEs in HK1 cells, suggesting these genes are bona fide SE-driven genes in SCC cells (Figure. S1C and Figure. S1D). Among these 91 SEs-driven genes, PDLIM1, ITGA6, PKPI, FAT2, and CAV2 genes, which are closely related to the occurrence and development of SCC, were selected for representative demonstration. As shown in Figure. S1E, ChIP-seq data analysis revealed that there were super enhancers marked by intensive and broad H3K27ac modifications at nearby the locus of PDLIM1, ITGA6, PKPI, FAT2, and CAV2 genes in HK1 and BxPC3 cells. We further analysis the expression levels of these genes in human SCC samples by analyzing the HNSC-TCGA database. The results showed that these genes' expression was significantly higher in SCC than in normal tissue (Figure. S1F), indicating that acquisition of SEs maybe the cause of abnormal overexpression of oncogenes in SCC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eJQ1/I-BET151 regulated the expression of ITGA6 together with SEs\u003c/h2\u003e \u003cp\u003eTo further evaluate the role of SEs in driving oncogene expression in SCC cells, we chose integrin α6 (ITGA6), also known as CD49f, as a representative of SEs-driven oncogene because it is a member of the integrin family and is associated with tumor growth, migration, and stemness(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). To assess the effect of the BRD4 inhibitor on ITGA6 expression in SCC, we treated HK1 and BxPC3 cells with different concentrations of JQ1 and I-BET.151. RT-qPCR and Western blot showed JQ1 and I-BET151 suppressed the mRNA and protein expression of ITGA6 in a dose-dependent manner (Figure. 2A and Figure. 2B). Using IGV software to analyze H3K27ac ChIP-seq data from HK1 and BxPC3 cells(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), we found there were four loci marked with high level of H3K27ac at SEs of ITGA6 (Figure. 2C). Then we employed CRISPR/Cas9-mediated enhancer repression to assess the crucial roles of SE in driving ITGA6 transcription. According to sequences of the four H3K27ac enrichment sites, we designed four sgRNAs and introduced these sgRNAs into dCas9-KRAB expressing HK1 cells. RT-qPCR and Western blot assays showed that when sgRNA guided transcription inhibitor KRAB to the H3K27ac enrichment site upstream of ITGA6, the transcription, and expression of ITGA6 was also inhibited (Figure. 2D and Figure. 2E), indicating that the expression of ITGA6 in SCC was regulated by SEs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eITGA6-SE had TP63 dependence\u003c/h2\u003e \u003cp\u003eOur previous study indicated that ΔNp63α, a prominent isoform of TP63(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), is the core factor enriched in SEs of HK1 cells(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Other studies also highlighted an essential role of ΔNp63α in shaping the SEs landscape in pancreatic squamous cell carcinoma(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), prompting us to consider whether SEs of ITGA6 were regulated by ΔNp63α. By analyzing the TCGA database of head and neck SCC, we found that ITGA6 and TP63 expression levels were positively correlated (Figure. 3A). RT-qPCR and Western blot assays showed that ITGA6 and TP63 were expressed in a variety of head and neck tumor cells. Furthermore, we found that ITGA6 was highly expressed in the cells with high ΔNp63α expression and ITGA6 was low expressed in the cells with low ΔNp63α expression (Figure. 3B and Figure. 3C). We used specific siRNAs to silence TP63 in HK1 and BxPC3 cells. RT-qPCR and Western blot assays demonstrated that transient transfection with TP63 siRNAs efficiently suppressed the mRNA and protein levels ITGA6 (Figure. 3D and 3E). Thus, our data suggested that there is a regulatory relationship between TP63 and ITGA6.\u003c/p\u003e \u003cp\u003eUsing IGV software to analyze TP63 and H3K27ac ChIP-seq data in BxPC3 cells, we found that TP63 protein was highly enriched at the ITGA6-SEs with H3K27ac modifications, whereas the levels of H3K27ac at ITGA6-SEs were dramatically decreased after TP63 knockout by CRISPR/Cas9 approach (Figure. 3F). To further confirm that the ITGA6-SE relies on TP63, we designed four primers according to the binding site. We used the histone acetyl-modified antibody anti-H3K27ac to perform ChIP-qPCR assays on TP63-deficiency cells, revealing that the levels of H3K27ac at SEs of ITGA6 were significantly decreased after TP63 knockdown (Figure. 3G). These results collectively support the notion that the overexpression of ITGA6 in SCC is driven by the SE, which is TP63-dependent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eITGA6 promoted the malignant progression of SCC cells\u003c/h2\u003e \u003cp\u003eWe further asked whether overexpression of ITGA6 contribute to acquisition of stemness features in SCC. ITGA6 expression was analyzed using GEPIA data to determine the role of ITGA6 in SCC development. ITGA6 was highly expressed in various squamous cell carcinomas (Figure. S2A). We analyzed the UALCAN database and found that ITGA6 expression was higher in head and neck SCC and pancreatic SCC samples than in normal control samples (Figure. S2B). Using KM-plot software to analyze the relationship between ITGA6 expression level and prognosis of patients with SCC, we found that patients with higher expression of ITGA6 had decreased overall survival (OS) (Figure. S2C), which indicated that the expression of ITGA6 was closely associated with prognosis of SCC. Moreover, the UALCAN database revealed that ITGA6 was associated with the grade of head and neck SCC and pancreatic SCC. The expression of ITGA6 increased with the deterioration of the tumor (Figure. S2D), but when the patient was in G4 grade, the expression of ITGA6 decreased significantly, while the mechanism was unclear.\u003c/p\u003e \u003cp\u003eWe examined the expression of ITGA6 in oral squamous carcinoma tissue samples, and the results of RT-qPCR and Western Blot showed that the expression of ITGA6 was higher in oral squamous carcinoma than in matched adjacent non-tumor tissues (Figure. 4A and Figure. 4B).To determine whether overexpression of ITGA6 contribute to acquisition of stemness features in SCC, we exploited shRNAs expressing lentivirus to silence ITGA6 expression. RT-qPCR and Western blot assays showed that two shRNAs targeted to ITGA6 effectively reduced its mRNA and protein levels in HK1 and BxPC3 cells (Figure. 4C and Figure. 4D). Tumor sphere formation assays showed that loss of ITGA6 weakened the sphere-forming ability of HK1 and BXPC3 cells (Figure. 4E), and RT-qPCR assays indicated that ITGA6-knockdown induced downregulation of CD133, CD44 and Snail expression in HK1, BxPC3 cells (Figure. 4F). Drug resistance is a feature of CSCs(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). We treated ITGA6 deficient HK1 and BxPC3 cells with cisplatin at different concentrations for chemotherapy sensitivity analysis. CCK8 assays revealed that inhibition of ITGA6 resulted in decreased cell viability (Figure. S3A).\u003c/p\u003e \u003cp\u003eIn addition, CCK8 assay showed that ITGA6 deficiency suppressed cell viability in HK1 and BxPC3 cells (Figure. S3B). The scratch wound-healing assays revealed that ITGA6 deficient SCC cells exhibited significantly reduced mobility compared with vector control cells (Figure. S3C). We also performed the Matrigel-coated Boyden chamber invasion assays, and the results demonstrated that ITGA6 deficiency suppressed SCC cell invasiveness in vitro (Figure. S3D). The expression of Ki67 (cell proliferation antigen) in cells was detected using immunofluorescence, which showed that the intensity of Ki67-positive cells decreased after stable interference with ITGA6, demonstrating that ITGA6 can promote the growth of SCC cells (Figure. S3E). Colony formation assays revealed that the loss of ITGA6 also reduced the colony numbers of HK1 and BxPC3 cells (Figure. S3F).\u003c/p\u003e \u003cp\u003eWe then asked whether ITGA6 promote SCC malignant behavior in vivo using a subcutaneous xenograft tumor model. Nude mice were injected with 0.1 ml containing 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e, 2.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e, 1.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e, 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e, and 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e tumor cells, respectively. Our data revealed that when the number of inoculated cells was \u0026ge;\u0026thinsp;2.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e/0.1 mL, the tumorigenic rate of HK1 cells was 5/5, while when the number of inoculated cells was \u0026le;\u0026thinsp;1.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e/0.1mL, the tumorigenic rate of ITGA6 deficient HK1 cells was lower than that of the control group, indicating that stable ITGA6 knockdown would lead to a decrease in the number of tumorigenic cells (Figure. 4G). At the endpoint of the experiment, we found xenografts from ITGA6 knockdown cells were much smaller than the vector control cells (Figure. 4H and Figure. 4I). Thus, these data collectively indicated that ITGA6 is closely related to stemness of SCC cells.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eITGA6 regulated the stemness of SCC cells through c-Myc active pathway\u003c/h2\u003e \u003cp\u003eTo address the downstream effectors of ITGA6 in regulating stemness of SCC, we employed RNA-seq analysis to screen transcriptomic alteration induced by loss of ITGA6. GSEA analysis revealed that the transcriptome in ITGA6\u003csup\u003ehigh\u003c/sup\u003e HK1 cells was positively correlated with c-Myc activity (Figure. 5A). c-Myc is a classical oncogene and a master regulator of cancer stem cells(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). By analyzing HNSCC-TCGA dataset, we found the expression of c-Myc was higher in SCC tissues than in normal control tissues and was associated with poor prognosis in tumor patients (Figure. S4A and Figure. S4B). Two c-Myc targeting shRNAs expressing lentivirus or an c-Myc cDNA-encoding lentivirus were introduced into HK1 and BxPC3 cells. Western blots assays revealed that two c-Myc targeting shRNAs successfully reduced c-Myc mRNA and protein levels, whereas c-Myc cDNA-encoding lentivirus upregulated c-Myc mRNA and protein levels in HK1 and BxPC3 cells (Figure. S4C). CCK8 assays showed that silencing c-Myc in HK1 and BxPC3 cells suppressed cell growth in vitro. In contrast, forced expression of c-Myc accelerated cell proliferation in SCC cells (Figure. S4D). RT-qPCR assays indicated that stable depletion of c-Myc led to decreased expression of cancer stem cell marker genes, including CD133, Snail. Correspondingly, stable depletion of c-Myc weakened the sphere-forming ability of HK1 and BxPC3 cells (Figure. S4E and Figure. S4F).\u003c/p\u003e \u003cp\u003eRT-qPCR assays showed that the mRNA levels of c-Myc in HK1 and BxPC cells were not affected by silencing ITGA6 (Figure. 5B). However, Western blot assays demonstrated that the protein levels of c-Myc were down-regulated after stable deficiency with ITGA6 in HK1 and BxPC3 cells (Figure. 5C). To verify that ITGA6 could promote the malignant progression of SCC by regulating c-Myc, we conducted functional rescue assays. We overexpressed c-Myc in ITGA6-deficient HK1 and BxPC3 cells using an c-Myc-expressing lentivirus. RT-qPCR and Western blot assays revealed that c-Myc -expressing lentivirus upregulated c-Myc mRNA and protein levels in HK1 and BxPC3 cells (Figure. 5D and Figure. 5E). CCK8 assays demonstrated that forced expression of c-Myc rescued cell proliferation in ITGA6 knockdown cells (Figure. 5F). RT-qPCR assays revealed that Snail expression was restored after overexpression of c-Myc in ITGA6 knockdown cells (Figure. 5G). Correspondingly, the sphere-forming ability of HK1 and BxPC3 cells was rescued by overexpression of c-Myc (Figure. 5H), suggesting that ITGA6 promotes stemness features of SCC cells through upregulating c-Myc protein.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eITGA6 regulated c-Myc protein through the ubiquitin-proteasome pathway\u003c/h2\u003e \u003cp\u003ec-Myc, as a classical oncogene, is most notably characterized by its short life span, instability, and susceptibility to degradation, with the ubiquitin-proteasome pathway being the predominant mechanism for c-Myc degradation(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). We analyzed the RNA-seq database and found that ITGA6 deficiency could upregulate FBXO32, which was also revealed by RT-qPCR and Western blot assays (Figure. 6A and Figure. 6B). FBXO32 is an F-box protein that combines SKP1, CUL1, and ROC-1 to form SCF-type E3 ubiquitin ligase(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). And what\u0026rsquo;s more, it has been reported that FBXO32 could target and degrade c-Myc protein through the proteasome pathway(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). HNSCC-TCGA data revealed that the expression of FBXO32 was lower in SCC tissues than in normal control tissues (Figure. 6C). We used specific siRNAs to silence FBXO32 in HK1 and BxPC3 cells. RT-qPCR and Western blot assays indicated that FBXO32-knockdown did not affect the mRNA levels of c-Myc but led to the upregulation of c-Myc protein levels. (Figure. 6D and Figure. 6E). Moreover, RT-qPCR and Western blot assays demonstrated that FBXO32 deficiency significantly rescued the protein levels of c-Myc in ITGA6 knockdown cells, with a small effect on its mRNA levels. (Figure. 6F and Figure. 6G). Correspondingly, tumor sphere formation assays showed that FBXO32 deficiency rescued the sphere-forming ability of ITGA6 knockdown cells (Figure. 6H).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eITGA6 regulated FBXO32 expression by regulating YAP1 entry into the nucleus\u003c/h2\u003e \u003cp\u003eTo address the mechanism by which ITGA6 regulated FBXO32, we performed further analysis of the RNA-seq database. GSEA analysis revealed that ITGA6 deficiency was positively correlated with YAP conserved signature (Figure. 7A). Yes-associated protein 1(YAP1) is the main effector of the Hippo pathway, which can cooperate with its transcriptional coactivator TAZ to promote cell proliferation, stem cell maintenance and tissue homeostasis (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Furthermore, YAP is related to SCC cells' deformation and metastasis potential (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Considering that the Hippo-YAP/TAZ pathway can be regulated by the cytoskeleton and is the main receptor of cells to mechanical forces(\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), we observed the changes in the mechanical characteristics of ITGA6 deficient cells. Using atomic force microscopy, we found that the cell surface roughness (Rq value) decreased after ITGA6 knockdown in HK1 and BxPC3 cells, indicating that the cell surface became smoother, resulting in reduced tumor motility (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). In addition, after stably knockdown ITGA6, the cell stiffness value increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), that is, the cell hardness increased, indicating that the cell deformation ability decreased, further proving that knockdown of ITGA6 can inhibit the migration and invasion of SCC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRT-qPCR and Western blot experiments showed that the knockdown of ITGA6 did not affect YAP1's mRNA and protein levels (Figure. 7D and Figure. 7E). Immunofluorescence assays showed that YAP1 protein was localized in the cytoplasm where it was colocalized with ITGA6 (Figure. S5A). Cytoplasmic and nucleus separation assays showed that ITGA6 deficiency could promote YAP1 entry into the nucleus (Figure. 7F). Western blot assays revealed that two siRNAs targeted to YAP1 effectively reduced their protein levels in HK1 and BxPC3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). We found that YAP1 co-knockdown could reverse the upregulation of FBXO32 expression induced by ITGA6 knockdown (Figure. 7H and Figure. 7I), suggesting induction of FBXO32 by loss of ITGA6 is mediated by YAP1. Furthermore, tumor sphere formation assays showed that co-knockdown of YAP1 could rescue the sphere-forming ability of ITGA6-knockdown cells (Figure. S5B). Correspondingly, RT-qPCR assays indicated that co-knockdown of YAP1 could rescue CD133, CD44, and Snail expression in ITGA6 deficient cells (Figure. S5C). CCK8 assays showed that co-knockdown of YAP1 in ITGA6 deficient cells could reverse the inhibition of cell viability (Figure. S5D). These data suggested that ITGA6 may enhance squamous carcinoma's stemness and malignant behavior by binding to YAP1 and inhibiting YAP1 entry into the nucleus.\u003c/p\u003e \u003cp\u003eThe transcriptional regulator YAP1 does not have a DNA-binding domain, and it uses the TEAD1 (TEA domain) family of transcription factors predominantly to elicit most of their biologically relevant gene expression programs(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). We used specific siRNAs to silence TEAD1 in HK1 and BxPC3 cells. RT-qPCR assays demonstrated that transfection with siRNAs efficiently suppressed the mRNA level of TEAD1, and the expression level of FBXO32 was downregulated after the TEAD1 knockdown in HK1, BxPC3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). ChIP-qPCR assays showed that TEAD1 binds to the promoter region of FBXO32(Figure.7K). Thus, these data suggested that ITGA6 could affect the malignant behavior of SCC cells through YAP1/TEAD1, FBXO32, and c-Myc axes.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we uncovered that overexpression of ITGA6 driven by TP63-dependent super enhancer enhances stemness features in SCC through regulating a YAP1-FBXO32-c-Myc axis, whereas disruption of super enhancer driven ITGA6 by BRD4 inhibitors suppresses stemness features in SCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Our study suggested targeting super enhancer may provide a therapeutic strategy for eliminating cancer stem cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEnhancers, a DNA sequence that can bind to transcription factors and enhance the transcription of target genes, play an important role in cell differentiation, body development, and tumorigenesis(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Activated enhancers are usually enriched for histone H3K4me1/2 and H3K27ac modifications (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). In 2013, American scientists proposed the concept of SEs (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Compared with ordinary enhancers, SEs can promote gene transcription more effectively(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Scientists have found that many key cancer genes' expressions are regulated by SEs. For example, in melanoma, researchers have found that SEs drive the high expression of AMIGO2, which promotes the malignant progression of tumors(\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Disruption of SEs often brings many changes to the cell. We found that JQ1 and I-BET151 treatment could reduce the stemness and inhibit the malignant behavior of SCC cells. These results suggest that cancer stemness genes may be regulated by SEs and the disruption of SEs is a promising approach to eliminate CSCs in SCC. This hypothesis is also supported by other\u0026rsquo;s observation that cancer stemness genes are regulated by SEs in SCC (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eWe identified 91 SEs driven genes in HK1 cells. According to the TCGA data, most of these SEs driven-genes are overexpressed in HNSCC samples and are associated with poor prognosis, highlighting super-enhancer-associated genes contribute to cancer progression(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Among them, ITGA6, as a member of integrin, participates in regulating various life activities, and its abnormal expression is related to the occurrence and development of tumors. Many studies have shown that ITGA6 is closely related to the malignant progression of tumors (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). In addition, ITGA6 expression has been detected in more than 30 stem cells, and ITGA6 has been identified as the only gene co-expressed in embryonic stem cells, embryonic neural stem cells, and hematopoietic stem cells(\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). These studies suggested that ITGA6 plays a very important role in stem cell biology. We provide strong evidence showing that ITGA6 is overexpressed in HNSCC samples and associated with unfavorable prognosis of HNSCC patients. Notably, CRISPR/Cas9-mediated enhancer repression confirmed that the expression of ITGA6 in SCC cells is tightly regulated by SEs. We also provide experimental evidence confirming that high expression of ITGA6 is required for maintenance of tumor initiating cells in SCC. Thus, ITGA6 is a bona fide SE-driven cancer stemness gene.\u003c/p\u003e \u003cp\u003eOur and other\u0026rsquo;s studies established an essential role of ΔNp63α in shaping SCC specific SEs landscape(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). In this study, we found that SEs at ITGA6 locus relies on ΔNp63α, because depletion of ΔNp63α by CRISPR/Cas9 or RNAi resulted in loss of H3K27ac modification at SEs of ITGA6 and consequent reduction of ITGA6 expression level. As the prominent isoform of TP63 in SCC, ΔNp63α is a well-recognized lineage survival oncogene (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Our study indicates that ΔNp63α may regulate cancer stemness genes by shaping SEs, which is in consistent with other\u0026rsquo;s observation that ΔNp63α contributes to stemness characteristics in SCC(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRNA-Seq analysis suggested that ITGA6\u003csup\u003ehigh\u003c/sup\u003e HK1 cells exhibit high c-Myc activity. c-Myc is a nuclear transcription factor which has a pivotal role in maintaining cancer stem cell populations(\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). One of the most prominent features of c-Myc is its short life span, instability, and easy degradation. Ubiquitin- proteasome pathway is the main mechanism of c-Myc degradation(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). In our study, we found that ITGA6 deficiency did not regulate the mRNA levels of c-Myc but caused a decrease in c-Myc protein levels. Overexpression of c-Myc in ITGA6 knockdown cells can restore the tumor sphere forming ability and the expression of CSCs markers in SCC cells. Accelerated c-Myc protein degradation in ITGA6 deficient cells is due to upregulation of FBXO32, a known E3 ligase targeting c-Myc for ubiquitin-proteasomal degradation(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). No evidence suggest ITGA6 could directly mediate gene transcription. Thus, upregulation of the mRNA level of FBXO32 upon inhibition of ITGA6 should be a consequence of altered signal transduction in ITGA6 deficient cells. GSEA analysis on transcriptomic alterations induced by loss of ITGA6 suggested YAP signaling involved. We found inhibition of ITGA6 led to increased YAP1 entry into the nucleus. We further provide evidence showing that FBXO32 is a direct target gene of YAP1/TEAD1 complex. Functionally, silencing YAP1 in ITGA6-deficient cells resulted in reduction of the level of FBXO32, but restoration of c-Myc protein and stemness features of SCC cells, indicating that ITGA6 sustains cancer stemness through preventing activation of YAP1/TEAD1-FBXO32 signaling.\u003c/p\u003e \u003cp\u003eMany previous studies have shown that hyper-activation of YAP signaling pathway promotes the expansion of cancer stem cells and drug resistance of cancer cells(\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). However, increasing evidence suggests that YAP1 plays a dual role in tumorigenesis and progression. Some scientists have found that cancers can be divided into YAP-on and YAP-off categories according to the presence or absence of YAP protein. For YAP-on cancers, cancer cells need YAP to grow and survive, while for YAP-off cancers, the presence of YAP can inhibit the growth of cancer cells (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). In esophageal squamous cell carcinoma, YAP1 functions as a tumor suppressor, and patients with a high YAP1 expression have better overall survival(\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). There are also controversies about roles of YAP1 in self-renewal vs. differentiation. It has been shown that in mouse embryonic stem cells, YAP1 is indispensable for self-renewal but required for differentiation. Nuclear translocation of YAP1 in mouse embryonic stem cells leads to disruption of self-renewal and triggering differentiation(\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). In this study, increase of nuclear translocation of YAP1 attenuated stemness features and malignant bechaviors of SCC cells upon loss of ITGA6, suggesting a tumor suppressive role of YAP1 in this context. We believe that whether YAP1 plays oncogenic or tumor suppressive roles in cancers may depend on the specific cellular contexts.\u003c/p\u003e \u003cp\u003eTo date, we don\u0026rsquo;t have a clear idea on how ITGA6 in regulating the nuclear translocation and activity of YAP1. It has been shown that phosphorylation of YAP by active LATS1/2 kinases causes sequestration of YAP in cytoplasm via 14-3-3 protein(\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). We observed that ITGA6 colocalized with YAP1 protein in cytoplasm of SCC cells. We speculate that ITGA6 may bind with YAP1 and exert a similar function as 14-3-3 protein. Another possibility is that increase of YAP1 nuclear translocation is triggered by alterations of the biomechanical properties of SCC cells upon silencing ITGA6, since the YAP/TAZ sense and respond to biomechanical signals(\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, our results demonstrate that BRD4 inhibitors suppresses stemness features in SCC cells. We identified a panel of SEs-driven genes associated with stemness features in SCC. Among these SEs-driven genes, ITGA6 is driven by ΔNp63α-dependent SEs and overexpressed in SCC. Elevated expression of ITGA6 stabilizes c-Myc protein via inhibition on YAP1/TEAD1-FBXO32 signal, which target c-Myc for degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Our study provides a new idea for epigenetically targeting CSCs.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003esquamous cell carcinoma (SCC); super enhancers (SEs); cancer stem cells (CSCs); Bromodomain Containing 4 (BRD4); Integrin \u0026alpha;6 (ITGA6); phosphate buffered saline (PBS); Cell Counting Kit-8 (CCK-8); atomic force microscope (AFM); Yes1 associated transcriptional regulator (YAP1); TEA domain transcription factor 1 (TEAD1); F-Box Protein 32 (FBXO32); Head and neck squamous cell carcinoma (HNSCC); The Cancer Genome Atlas (TCGA); clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproval for the use of clinical samples and information was obtained from each patient and the Research Ethics Committee of Central South University. Written informed consent was received prior to patient participation. The animal experiments were conducted according to the protocol approved by the Animal Welfare Committee of Central South University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read the manuscript\u0026nbsp;and agree to publish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eThe datasets and materials used and/or analyzed during the current study are available\u0026nbsp;from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no conflict of interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported in part by grants from The National Natural Science\u0026nbsp;Foundation of China (82272631,\u0026nbsp;82072596, 82173339, 82172766, 81872278), the National\u0026nbsp;\u0026ldquo;111\u0026rdquo; Project (Project #111-2-12), the Hunan Provincial Key Research\u0026nbsp;and Development Program (2022SK2026), the Natural Science Foundation of Hunan\u0026nbsp;Province, China (2020JJ4920, 2020JJ4838, 2020JJ4766, 2020JJ3055), the Scientific Research Project of Hunan Provincial Health Commission (20201067, 20201040), the Beijing Xisike Clinical Oncology Research Foundation (Y-HR2020ZD-0052), the Open Funds of State Key Laboratory of Oncology in south China (HN2021\u0026ndash;07). We thank our colleagues\u0026nbsp;at the cancer research\u0026nbsp;institute of central south university for constructive discussion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBo Xiang and Mei Yi designed research. Ying Liu, Meng Hu, Jing Cai and Quanzhu Chen performed most of the experiments and prepared figures 1-8. Mei Yi and Bo Xiang analyzed the data. Mei Yi and Bo Xiang wrote the main manuscript text. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSong Q, Yang Y, Jiang D, Qin Z, Xu C, Wang H, et al. Proteomic analysis reveals key differences between squamous cell carcinomas and adenocarcinomas across multiple tissues. Nat Commun. 2022;13(1):4167.\u003c/li\u003e\n\u003cli\u003eYi M, Tan Y, Wang L, Cai J, Li X, Zeng Z, et al. 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Nat Cell Biol. 2018;20(8):888-99.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Integrin, Cancer stem cells, Super-enhancer, ITGA6, Myc, TP63","lastPublishedDoi":"10.21203/rs.3.rs-3017766/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3017766/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSquamous cell carcinoma (SCC) is life-threatening malignancy. Cancer stem cells (CSCs) are associated with SCCs initiation, metastasis, therapy resistance, and relapse. Acquisition of super enhancer is a cause of hyper-activation of oncogenes in cancer, contributing to tumorigenesis and progression. In this study, we found disruption of SEs-associated transcription by BRD4 inhibitors JQ1 and I-BET151 inhibited the stemness features of SCC cells. Combined analysis with transcriptomics alterations induced by treatments of BRD4 inhibitors and SEs profile of SCC cells identified SEs-driven oncogenes in SCC cells. Among these SEs-driven oncogenes, ITGA6 (Integrin subunit α6) was highly expressed in SCC samples and predicted unfavorable prognosis in SCC patients. The ΔNp63α, a lineage-survival oncogene in SCC, enriched at ITGA6-SEs and was responsible for the activation of ITGA6-SEs. Silencing of ITGA6 substantially impeded the stemness features in vitro, as well as reduced thenumber of tumor-initiating cells of SCC in vivo. Mechanistically, silencing of ITGA6 resulted in the degradation of c-Myc protein via upregulation of an E3 ubiquitin ligase FBXO32. Furthermore, we demonstrated silencing of ITGA6 promoted nuclear translocation of YAP1, which facilitated TEAD1-mediated transcription of FBXO32 in SCC cells. Thus, our data suggested ITGA6 contributes to maintaining stemness features of SCC through a YAP1/FBXO32/c-myc cascade, providing a therapeutic target for eliminating cancer stem cells.\u003c/p\u003e","manuscriptTitle":"Super-enhancer-driven ITGA6 enhances stemness features of squamous cell carcinoma through stabilizing c-myc protein","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-11 14:45:09","doi":"10.21203/rs.3.rs-3017766/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fa6b6d96-23d8-4dd9-af17-44d872425743","owner":[],"postedDate":"July 11th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":23047560,"name":"Biological sciences/Cancer/Cancer stem cells"},{"id":23047561,"name":"Biological sciences/Cancer/Head and neck cancer"}],"tags":[],"updatedAt":"2024-08-11T11:29:49+00:00","versionOfRecord":[],"versionCreatedAt":"2023-07-11 14:45:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3017766","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3017766","identity":"rs-3017766","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}