A refined Uni-vector prime editing system improves genome editing outcomes in mammalian cells.

Prime editing is an advanced technology in CRISPR/Cas research with increasing numbers of improved methodologies. The original multi-vector method hampers the efficiency and precision of prime editing and also has inherent difficulty in generating homozygous mutations in mammalian cells. To overcome these technical issues, we developed a Uni-vector prime editing system, wherein the major components for prime editing were constructed in all-in-one plasmids, pPE3-pPuro and pePEmax-pPuro. The Uni-vector prime editing plasmids enhance the editing efficiency of prime editing and improved the generation of homozygous mutated mammalian cell lines. The editing efficiency is dependent of the transfection efficiency. Remarkably, the Uni-vector ePE5max system achieved an impressive editing rate approximately 79% in average, even in cell lines that are traditionally difficult to transfect, such as FaDu cell line. Furthermore, it resulted in a high frequency of homozygous knocked-in cells, with a rate of 99% in HeLa and 85% in FaDu cells. Together, our Uni-vector approach simplifies the delivery of editing components and improves the editing efficiency, especially in cells with low transfection efficiency. This approach presents an advancement in the field of prime editing.

PLK1 and its substrate MISP facilitate intrahepatic cholangiocarcinoma progression by promoting lymphatic invasion and impairing E-cadherin adherens junctions.

Intrahepatic cholangiocarcinoma (iCCA) is a subtype of CCA and has a high mortality rate and a relatively poor prognosis. However, studies focusing on increased cell motility and loss of epithelial integrity during iCCA progression remain relatively scarce. We collected seven fresh tumor samples from four patients to perform RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) to determine the transcriptome profile and chromatin accessibility of iCCA. The increased expression of cell cycle regulators, including PLK1 and its substrate MISP, was identified. Ninety-one iCCA patients were used to validate the clinical significance of PLK1 and MISP. The upregulation of PLK1 and MISP was determined in iCCA tissues. Increased expression of PLK1 and MISP was significantly correlated with tumor number, N stage, and lymphatic invasion in an iCCA cohort. Knockdown of PLK1 or MISP reduced trans-lymphatic endothelial migration and wound healing and affected focal adhesions in vitro. In cell‒cell junctions, MISP localized to adherens junctions and suppressed E-cadherin dimerization. PLK1 disrupted adherens junctions in a myosin-dependent manner. Furthermore, PLK1 and MISP promoted cell proliferation in vitro and tumorigenesis in vivo. In iCCA, PLK1 and MISP promote aggressiveness by increasing lymphatic invasion, tumor growth, and motility through the repression of E-cadherin adherens junctions.

Epigenetic regulation of asymmetric cell division by the LIBR-BRD4 axis.

Asymmetric cell division (ACD) is a mechanism used by stem cells to maintain the number of progeny. However, the epigenetic mechanisms regulating ACD remain elusive. Here we show that BRD4, a BET domain protein that binds to acetylated histone, is segregated in daughter cells together with H3K56Ac and regulates ACD. ITGB1 is regulated by BRD4 to regulate ACD. A long noncoding RNA (lncRNA), LIBR (LncRNA Inhibiting BRD4), decreases the percentage of stem cells going through ACD through interacting with the BRD4 mRNAs. LIBR inhibits the translation of BRD4 through recruiting a translation repressor, RCK, and inhibiting the binding of BRD4 mRNAs to polysomes. These results identify the epigenetic regulatory modules (BRD4, lncRNA LIBR) that regulate ACD. The regulation of ACD by BRD4 suggests the therapeutic limitation of using BRD4 inhibitors to treat cancer due to the ability of these inhibitors to promote symmetric cell division that may lead to tumor progression and treatment resistance.

RP11-367G18.1 V2 enhances clear cell renal cell carcinoma progression via induction of epithelial-mesenchymal transition.

PURPOSE: Metastasis is the end stage of renal cell carcinoma (RCC), and clear cell renal cell carcinoma (ccRCC) is the most common malignant subtype. The hypoxic microenvironment is a common feature in ccRCC and plays an essential role in the regulation of epithelial-mesenchymal transition (EMT). Accumulating evidence manifests that long non-coding RNAs (lncRNAs) participate in RCC tumorigenesis and regulate hypoxia-induced EMT. Here, we identified a lncRNA RP11-367G18.1 induced by hypoxia, that was overexpressed in ccRCC tissues. METHODS: A total of 216 specimens, including 149 ccRCC tumor samples and 67 related normal kidney parenchyma tissue samples, were collected. To investigate the biological fucntions of RP11.367G18.1 in ccRCC, migration, invasion, soft agar colony formation, xenograft tumorigenicity assays, and tail vein and orthotopic metastatic mouse models were performed. The relationship between RP11-367G18.1 and downstream signaling was analyzed utilizing reporter assay, RNA pull-down, chromatin immunopreciptation, and chromatin isolation by RNA purification assays. RESULTS: Hypoxic conditions and overexpression of HIF-1α increased the level of RP11-367G18.1. RP11-367G18.1 induced EMT and enhanced cell migration and invasion through variant 2. Inhibition of RP11-367G18.1 variant 2 reversed hypoxia-induced EMT phenotypes. An in vivo study revealed that RP11-367G18.1 variant 2 was required for hypoxia-induced tumor growth and metastasis in ccRCC. Mechanistically, RP11-367G18.1 variant 2 interacted with p300 histone acetyltransferase to regulate lysine 16 acetylation on histone 4 (H4K16Ac), thus contributing to hypoxia-regulated gene expression. Clinically, RP11-367G18.1 variant 2 was upregulated in ccRCC tissues, particularly metastatic ccRCC tissues, and it is linked to poor overall survival. CONCLUSION: These findings demonstrate the prognostic value and EMT-promoting role of RP11-367G18.1 and indicate that this lncRNA may provide a therapeutic target for ccRCC.

Organ defects of the Usp7K444R mutant mouse strain indicate the essential role of K63-polyubiquitinated Usp7 in organ formation.

BACKGROUND: K63-linked polyubiquitination of proteins have nonproteolytic functions and regulate the activity of many signal transduction pathways. USP7, a HIF1α deubiquitinase, undergoes K63-linked polyubiquitination under hypoxia. K63-polyubiquitinated USP7 serves as a scaffold to anchor HIF1α, CREBBP, the mediator complex, and the super elongation complex to enhance HIF1α-induced gene transcription. However, the physiological role of K63-polyubiquitinated USP7 remains unknown. METHODS: Using a Usp7K444R point mutation knock-in mouse strain, we performed immunohistochemistry and standard molecular biological methods to examine the organ defects of liver and kidney in this knock-in mouse strain. Mechanistic studies were performed by using deubiquitination, immunoprecipitation, and quantitative immunoprecipitations (qChIP) assays. RESULTS: We observed multiple organ defects, including decreased liver and muscle weight, decreased tibia/fibula length, liver glycogen storage defect, and polycystic kidneys. The underlying mechanisms include the regulation of protein stability and/or modulation of transcriptional activation of several key factors, leading to decreased protein levels of Prr5l, Hnf4α, Cebpα, and Hnf1ß. Repression of these crucial factors leads to the organ defects described above. CONCLUSIONS: K63-polyubiquitinated Usp7 plays an essential role in the development of multiple organs and illustrates the importance of the process of K63-linked polyubiquitination in regulating critical protein functions.

METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets.

BACKGROUND: DNA N6-methyldeoxyadenosine (6mA) is rarely present in mammalian cells and its nuclear role remains elusive. RESULTS: Here we show that hypoxia induces nuclear 6mA modification through a DNA methyltransferase, METTL4, in hypoxia-induced epithelial-mesenchymal transition (EMT) and tumor metastasis. Co-expression of METTL4 and 6mA represents a prognosis marker for upper tract urothelial cancer patients. By RNA sequencing and 6mA chromatin immunoprecipitation-exonuclease digestion followed by sequencing, we identify lncRNA RP11-390F4.3 and one novel HIF-1α co-activator, ZMIZ1, that are co-regulated by hypoxia and METTL4. Other genes involved in hypoxia-mediated phenotypes are also regulated by 6mA modification. Quantitative chromatin isolation by RNA purification assay shows the occupancy of lncRNA RP11-390F4.3 on the promoters of multiple EMT regulators, indicating lncRNA-chromatin interaction. Knockdown of lncRNA RP11-390F4.3 abolishes METTL4-mediated tumor metastasis. We demonstrate that ZMIZ1 is an essential co-activator of HIF-1α. CONCLUSIONS: We show that hypoxia results in enriched 6mA levels in mammalian tumor cells through METTL4. This METTL4-mediated nuclear 6mA deposition induces tumor metastasis through activating multiple metastasis-inducing genes. METTL4 is characterized as a potential therapeutic target in hypoxic tumors.

Genetic Disruption of KLF1 K74 SUMOylation in Hematopoietic System Promotes Healthy Longevity in Mice.

The quest for rejuvenation and prolonged lifespan through transfusion of young blood has been studied for decades with the hope of unlocking the mystery of the key substance(s) that exists in the circulating blood of juvenile organisms. However, a pivotal mediator has yet been identified. Here, atypical findings are presented that are observed in a knockin mouse model carrying a lysine to arginine substitution at residue 74 of Krüppel-like factor 1 (KLF1/EKLF), the SUMOylation-deficient Klf1K74R/K74R mouse, that displayed significant improvement in geriatric disorders and lifespan extension. Klf1K74R/K74R mice exhibit a marked delay in age-related physical performance decline and disease progression as evidenced by physiological and pathological examinations. Furthermore, the KLF1(K74R) knockin affects a subset of lymphoid lineage cells; the abundance of tumor infiltrating effector CD8+ T cells and NKT cells is increased resulting in antitumor immune enhancement in response to tumor cell administration. Significantly, infusion of hematopoietic stem cells (HSCs) from Klf1K74R/K74R mice extends the lifespan of the wild-type mice. The Klf1K74R/K74R mice appear to be an ideal animal model system for further understanding of the molecular/cellular basis of aging and development of new strategies for antiaging and prevention/treatment of age-related diseases thus extending the healthspan as well as lifespan.

Chromatin accessibility analysis identifies GSTM1 as a prognostic marker in human glioblastoma patients.

BACKGROUND: Glioblastoma (GBM) is a malignant human brain tumor that has an extremely poor prognosis. Classic mutations such as IDH (isocitrate dehydrogenase) mutations, EGFR (epidermal growth factor receptor) alternations, and MGMT (O6-methylguanine-methyltransferase) promoter hypermethylation have been used to stratify patients and provide prognostic significance. Epigenetic perturbations have been demonstrated in glioblastoma tumorigenesis. Despite the genetic markers used in the management of glioblastoma patients, new biomarkers that could predict patient survival independent of known biomarkers remain to be identified. METHODS: ATAC-seq (assay for transposase accessible chromatin followed by sequencing) and RNA-seq have been used to profile chromatin accessible regions using glioblastoma patient samples with short-survival versus long-survival. Cell viability, cell cycle, and Western blot analysis were used to characterize the cellular phenotypes and identify signaling pathways. RESULTS: Analysis of chromatin accessibility by ATAC-seq coupled with RNA-seq methods identified the GSTM1 (glutathione S-transferase mu-1) gene, which featured higher chromatin accessibility in GBM tumors with short survival. GSTM1 was confirmed to be a significant prognostic marker to predict survival using a different GBM patient cohort. Knockdown of GSTM1 decreased cell viability, caused cell cycle arrest, and decreased the phosphorylation levels of the NF-kB (nuclear factor kappa B) p65 subunit and STAT3 (signal transducer and activator of transcription 3) (pSer727). CONCLUSIONS: This report demonstrates the use of ATAC-seq coupled with RNA-seq to identify GSTM1 as a prognostic marker of GBM patient survival. Activation of phosphorylation levels of NF-kB p65 and STAT3 (pSer727) by GSTM1 is shown. Analysis of chromatin accessibility in patient samples could generate an independent biomarker that can be used to predict patient survival.

The role of hypoxia-induced long noncoding RNAs (lncRNAs) in tumorigenesis and metastasis.

Long noncoding RNAs (lncRNAs) are noncoding RNAs with length greater than 200 nt. The biological roles and mechanisms mediated by lncRNAs have been extensively investigated. Hypoxia is a proven microenvironmental factor that promotes solid tumor metastasis. Epithelial-mesenchymal transition (EMT) is one of the major mechanisms induced by hypoxia to contribute to metastasis. Many lncRNAs have been shown to be induced by hypoxia and their roles have been delineated. In this review, we focus on the hypoxia-inducible lncRNAs that interact with protein/protein complex and chromatin/epigenetic factors, and the mechanisms that contribute to metastasis. The role of a recently discovered lncRNA RP11-390F4.3 in hypoxia-induced EMT is discussed. Whole genome approaches to delineating the association between lncRNAs and histone modifications are discussed. Other topics related to hypoxia-induced tumor progression but require further investigation are also mentioned. The clinical significance and treatment strategy targeted against lncRNAs are discussed. The review aims to identify suitable lncRNA targets that may provide feasible therapeutic venues for hypoxia-involved cancers.

USP7 facilitates SMAD3 autoregulation to repress cancer progression in p53-deficient lung cancer.

USP7, one of the most abundant ubiquitin-specific proteases (USP), plays multifaceted roles in many cellular events, including oncogenic pathways. Accumulated studies have suggested that USP7, through modulating the MDM2/MDMX-p53 pathway, is a promising target for cancer treatment; however, little is known about the function of USP7 in p53-deficient tumors. Here we report that USP7 regulates the autoregulation of SMAD3, a key regulator of transforming growth factor ß (TGFß) signaling, that represses the cell progression of p53-deficient lung cancer. CRISPR/Cas9-mediated inactivation of USP7 in p53-deficient lung cancer H1299 line resulted in advanced cell proliferation in vitro and in xenograft tumor in vivo. Genome-wide analyses (ChIP-seq and RNA-seq) of USP7 KO H1299 cells reveal a dramatic reduction of SMAD3 autoregulation, including decreased gene expression and blunted function of associated super-enhancer (SE). Furthermore, biochemical assays show that SMAD3 is conjugated by mono-ubiquitin, which negatively regulates the DNA-binding function of SMAD3, in USP7 KO cells. In addition, cell-free and cell-based analyses further demonstrate that the deubiquitinase activity of USP7 mediates the removal of mono-ubiquitin from SMAD3 and facilitates the DNA-binding of SMAD3-SMAD4 dimer at SMAD3 locus, and thus enhance the autoregulation of SMAD3. Collectively, our study identified a novel mechanism by which USP7, through catalyzing the SMAD3 de-monoubiquitination, facilitates the positive autoregulation of SMAD3, and represses the cancer progression of p53-deficient lung cancer.

Liquid-liquid phase separation (LLPS) in cellular physiology and tumor biology.

Liquid-liquid phase separation (LLPS) has emerged as a mechanism that has been used to explain the formation of known organelles (e.g. nucleoli, promyelocytic leukemia nuclear bodies (PML NBs), etc) as well as other membraneless condensates (e.g. nucleosome arrays, DNA damage foci, X-chromosome inactivation (XCI) center, paraspeckles, stress granules, proteasomes, autophagosomes, etc). The formation of membraneless condensates could be triggered by proteins containing modular domains or intrinsically disordered regions (IDRs) and nucleic acids. Multiple biological processes including transcription, chromatin organization, X-chromosome inactivation (XCI), DNA damage, tumorigenesis, autophagy, etc have been shown to utilize the principle of LLPS to facilitate these processes. This review will summarize the principle and components of LLPS, and describe how LLPS regulate these numerous biological processes and disruption of LLPS would cause disease formation. The role of LLPS in regulating normal cellular physiology and contributing to tumorigenesis will be discussed.

Induction of epithelial-mesenchymal transition (EMT) by hypoxia-induced lncRNA RP11-367G18.1 through regulating the histone 4 lysine 16 acetylation (H4K16Ac) mark.

Hypoxia activates various long noncoding RNAs (lncRNAs) to induce the epithelial-mesenchymal transition (EMT) and tumor metastasis. The hypoxia/HIF-1α-regulated lncRNAs that also regulate a specific histone mark and promote EMT and metastasis have not been identified. We performed RNA-sequencing dataset analysis to search for such lncRNAs and lncRNA RP11-367G18.1 was the hypoxia-induced lncRNA with the highest hazard ratio. High expression of lncRNA RP11-367G18.1 is correlated with a worse survival of head and neck cancer patients. We further showed that lncRNA RP11-367G18.1 is induced by hypoxia and directly regulated by HIF-1α in cell lines. Overexpression of lncRNA RP11-367G18.1 induces the EMT and increases the in vitro migration and invasion and in vivo metastatic activity. Knockdown experiments showed that lncRNA RP11-367G18.1 plays an essential role in hypoxia-induced EMT. LncRNA RP11-367G18.1 specifically regulates the histone 4 lysine 16 acetylation (H4K16Ac) mark that is located on the promoters of two "core" EMT regulators, Twist1 and SLUG, and VEGF genes. These results indicate that lncRNA RP11-367G18.1 regulates the deposition of H4K16Ac on the promoters of target genes to activate their expression. This report identifies lncRNA RP11-367G18.1 as a key player in regulating the histone mark H4K16Ac through which activates downstream target genes to mediate hypoxia-induced EMT.

The epigenetic roles of DNA N6-Methyladenine (6mA) modification in eukaryotes.

The DNA N6-methyladenine (6mA) modification is a prevalent epigenetic mark in prokaryotes, but the low abundance of 6mA in eukaryotes has recently received attention. The possible role of 6mA as an epigenetic mark in eukaryotes is starting to be recognized. This review article addresses the epigenetic roles of 6mA in eukaryotes. The existence of 6mA in metazoans and plants, the correlation of 6mA with gene expression, the enzymes catalyzing the deposition and removal of the 6mA modification, the relationship of 6mA to nucleosome positioning, the 6mA interaction with chromatin, its role in tumorigenesis and other physiological conditions/diseases and technical issues in 6mA detection/profiling and bioinformatics analysis are described. New directions and unresolved issues (e.g., the base-pair-resolution 6mA-sequencing method and gene activation vs. repression) in 6mA research are discussed.

The role of miRNA biogenesis and DDX17 in tumorigenesis and cancer stemness.

Cancer stemness represents one of the major mechanisms that predispose patients to tumor aggressiveness, metastasis, and treatment resistance. MicroRNA biogenesis is an important process controlling miRNA processing and maturation. Deregulation of miRNA biogenesis can lead to tumorigenesis and cancer stemness. DDX17 is a co-factor of the miRNA microprocessor. Misregulation of DDX17 can be associated with cancer stemness. K63-linked polyubiquitination of DDX17 presents a concerted mechanism of decreased synthesis of stemness-inhibiting miRNAs and increased transcriptional activation of stemness-related gene expression. K63-linked polyubiquitination of HAUSP serves as a scaffold to anchor HIF-1α, CBP, the mediator complex, and the super-elongation complex to enhance HIF-1α-induced gene transcription. Recent progress in RNA modifications shows that RNA N6-methyladenosine (m6A) modification is a crucial mechanism to regulate RNA levels. M6A modification of miRNAs can also be linked to tumorigenesis and cancer stemness. Overall, miRNA biogenesis and K63-linked polyubiquitination of DDX17 play an important role in the induction of cancer stemness. Delineation of the mechanisms and identification of suitable targets may provide new therapeutic options for treatment-resistant cancers.

Hypoxia-induced lncRNA RP11-390F4.3 promotes epithelial-mesenchymal transition (EMT) and metastasis through upregulating EMT regulators.

Hypoxia-induced long noncoding RNAs (lncRNAs) have been shown to induce tumor metastasis. However, lncRNAs that are regulated by hypoxia/HIF-1α and subsequently control the expression of multiple epithelial-mesenchymal transition (EMT) regulators have not been identified. To identify such lncRNAs, analysis of RNA-sequencing datasets was performed. The lncRNA RP11-390F4.3 was shown to be induced by hypoxia and directly activated by HIF-1α. Overexpression of lncRNA RP11-390F4.3 induced EMT and metastasis. LncRNA RP11-390F4.3 was essential for hypoxia-induced EMT and metastasis. LncRNA RP11-390F4.3 overexpression induced the expression of multiple EMT regulators. This report demonstrates that LncRNA RP11-390F4.3 is induced by hypoxia/HIF-1α and is essential for hypoxia-induced EMT and metastasis via the activation of multiple EMT regulators.

Epigenetic regulation of epithelial-mesenchymal transition: focusing on hypoxia and TGF-ß signaling.

Epithelial-mesenchymal transition (EMT) is an important process triggered during cancer metastasis. Regulation of EMT is mostly initiated by outside signalling, including TGF-ß, growth factors, Notch ligand, Wnt, and hypoxia. Many signalling pathways have been delineated to explain the molecular mechanisms of EMT. In this review, we will focus on the epigenetic regulation of two critical EMT signalling pathways: hypoxia and TGF-ß. For hypoxia, hypoxia-induced EMT is mediated by the interplay between chromatin modifiers histone deacetylase 3 (HDAC3) and WDR5 coupled with the presence of histone 3 lysine 4 acetylation (H3K4Ac) mark that labels the promoter regions of various traditional EMT marker genes (e.g. CDH1, VIM). Recently identified new hypoxia-induced EMT markers belong to transcription factors (e.g. SMO, GLI1) that mediate EMT themselves. For TGF-ß-induced ΕΜΤ, global chromatin changes, removal of a histone variant (H2A.Z), and new chromatin modifiers (e.g. UTX, Rad21, PRMT5, RbBP5, etc) are identified to be crucial for the regulation of both EMT transcription factors (EMT-TFs) and EMT markers (EMT-Ms). The epigenetic mechanisms utilized in these two pathways may serve as good model systems for other signalling pathways and also provide new potential therapeutic targets.

N6-Deoxyadenosine Methylation in Mammalian Mitochondrial DNA.

N6-Methyldeoxyadenosine (6mA) has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA (gDNA). However, the biological functions of 6mA in mammals have yet to be adequately explored, largely due to its low abundance in most mammalian genomes. Here, we report that mammalian mitochondrial DNA (mtDNA) is enriched for 6mA. The level of 6mA in HepG2 mtDNA is at least 1,300-fold higher than that in gDNA under normal growth conditions, corresponding to approximately four 6mA modifications on each mtDNA molecule. METTL4, a putative mammalian methyltransferase, can mediate mtDNA 6mA methylation, which contributes to attenuated mtDNA transcription and a reduced mtDNA copy number. Mechanistically, the presence of 6mA could repress DNA binding and bending by mitochondrial transcription factor (TFAM). Under hypoxia, the 6mA level in mtDNA could be further elevated, suggesting regulatory roles for 6mA in mitochondrial stress response. Our study reveals DNA 6mA as a regulatory mark in mammalian mtDNA.

Identification of new hypoxia-regulated epithelial-mesenchymal transition marker genes labeled by H3K4 acetylation.

Hypoxia-induced epithelial-mesenchymal transition (EMT) involves the interplay between chromatin modifiers histone deacetylase 3 (HDAC3) and WDR5. The histone mark histone 3 lysine 4 acetylation (H3K4Ac) is observed in the promoter regions of various EMT marker genes (eg, CDH1 and VIM). To further define the genome-wide location of H3K4Ac, a chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) analysis was performed using a head and neck squamous cell carcinoma (HNSCC) FaDu cell line under normoxia and hypoxia. H3K4Ac was found to be located mainly around the transcription start site. Coupled with analysis of gene expression by RNA sequencing and using a HDAC3 knockdown cell line, 10 new genes (BMI1, GLI1, SMO, FOXF1, SIRT2, etc) that were labeled by H3K4Ac and regulated by HDAC3 were identified. Overexpression or knockdown of GLI1/SMO increased or repressed the in vitro migration and invasion activity in OECM-1/FaDu cells, respectively. In HNSCC patients, coexpression of GLI1 and SMO in primary tumors correlated with metastasis. Our results identify new EMT marker genes that may play a significant role in hypoxia-induced EMT and metastasis and further provide diagnostic and prognostic implications.

Author Correction: Bmi1 is essential in Twist1-induced epithelial-mesenchymal transition.

In the version of Supplementary Fig. 3c originally published with this Article, the authors mistakenly duplicated a blot from Supplementary Fig. 3b. The correct versions of these figures are shown below. In addition, two independent repeats of the experiments presented in Supplementary Figs. 3b and 3c, showing results consistent with those originally reported, have been deposited in Figshare ( 10.6084/m9.figshare.7545263 ).

Regulation of miRNA Biogenesis and Histone Modification by K63-Polyubiquitinated DDX17 Controls Cancer Stem-like Features.

Markers of cancer stemness predispose patients to tumor aggressiveness, drug and immunotherapy resistance, relapse, and metastasis. DDX17 is a cofactor of the Drosha-DGCR8 complex in miRNA biogenesis and transcriptional coactivator and has been associated with cancer stem-like properties. However, the precise mechanism by which DDX17 controls cancer stem-like features remains elusive. Here, we show that the E3 ligase HectH9 mediated K63-polyubiquitination of DDX17 under hypoxia to control stem-like properties and tumor-initiating capabilities. Polyubiquitinated DDX17 disassociated from the Drosha-DGCR8 complex, leading to decreased biogenesis of anti-stemness miRNAs. Increased association of polyubiquitinated DDX17 with p300-YAP resulted in histone 3 lysine 56 (H3K56) acetylation proximal to stemness-related genes and their subsequent transcriptional activation. High expression of HectH9 and six stemness-related genes (BMI1, SOX2, OCT4, NANOG, NOTCH1, and NOTCH2) predicted poor survival in patients with head and neck squamous cell carcinoma and lung adenocarcinoma. Our findings demonstrate that concerted regulation of miRNA biogenesis and histone modifications through posttranslational modification of DDX17 underlies many cancer stem-like features. Inhibition of DDX17 ubiquitination may serve as a new therapeutic venue for cancer treatment. SIGNIFICANCE: Hypoxia-induced polyubiquitination of DDX17 controls its dissociation from the pri-miRNA-Drosha-DCGR8 complex to reduce anti-stemness miRNA biogenesis and association with YAP and p300 to enhance transcription of stemness-related genes.