Hypoxia-induced DTL promotes the proliferation, metastasis, and sorafenib resistance of Hepatocellular Carcinoma through Notch Signaling Pathway

Hypoxia-induced DTL promotes the proliferation, metastasis, and sorafenib resistance of Hepatocellular Carcinoma through Notch Signaling Pathway | 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 Hypoxia-induced DTL promotes the proliferation, metastasis, and sorafenib resistance of Hepatocellular Carcinoma through Notch Signaling Pathway Fei Gao, Zi-Xiong Chen, Mao-Yuan Mu, Guang Yang, Han Qi, Xiao-Bo Fu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3691309/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Oct, 2024 Read the published version in Cell Death & Disease → Version 1 posted 12 You are reading this latest preprint version Abstract Denticleless E3 ubiquitin protein ligase homolog (DTL), the substrate receptor of the CRL4A complex, plays a central role in genome stability. Even though the oncogenic function of DTL has been investigated in several cancers, its specific role in Hepatocellular Carcinoma (HCC) still needs further elucidation. Data from a clinical cohort (n = 209), RNA-sequencing, and public database (TCGA and GEO) were analyzed, indicating that DTL is closely related to patient prognosis and could serve as a promising prognostic indicator in HCC. Functionally, DTL promoted the proliferation, metastasis, and sorafenib resistance of HCC in vitro. In the orthotopic tumor transplantation and tail vein injection model, DTL promoted the growth and metastasis of HCC in vivo. Mechanically, we revealed for the first time that DTL was transcriptionally activated by hypoxia-inducible factor 1α (HIF-1α) under hypoxia and functioned as a downstream effector molecule of HIF-1α. DTL facilitates HCC cell proliferation, metastasis, and epithelial-mesenchymal transition through the Notch pathway. These results suggested that DTL may be a potential biomarker and therapeutic target for HCC. Biological sciences/Cancer/Oncogenes Biological sciences/Cancer/Gastrointestinal cancer/Liver cancer Biological sciences/Cancer/Metastasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction With the fastest-increasing mortality for decades, liver cancer ranks as the third largest cancer-related death worldwide 1 , 2 . Hepatocellular carcinoma (HCC) accounts for ~ 90% of primary liver cancer, with a 5-year relative survival rate of approximately 18% 2 . Moreover, two-thirds of HCC patients are diagnosed at advanced stages with limited treatment options and worse prognosis 3 . Whilst our understanding of the molecular pathogenesis of HCC has been improved, few effective molecular targets or biomarkers have been translated into clinical practice 4 . Therefore, there is an urgent need to further understand the mechanisms underlying the HCC progression. Hypoxia, as the primary characteristic of the tumor microenvironment, plays a vital role in the progression of HCC 5 , 6 . As a hypermetabolic tumor, the uninhibited proliferation and abnormal formation of micro-vessels make HCC more susceptible to hypoxia 6 . Hypoxia-inducible transcription factor 1α (HIF-1α), the primary regulator of cellular adaptive responses to hypoxia, extensively participates in the malignant progression and resistance to chemotherapy and immunotherapy of HCC 7 , 8 . In the absence of oxygen, HIF-1α binds to hypoxia-responsive elements (HREs) and enhances transcription of downstream target genes 9 . The representative cancer-related genes regulated by HIF-1α include the vascular endothelial growth factor (VEGF), transforming growth factor (TGF-β), and P53 10 . Cullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases, are essential for many eukaryotic biological processes, including cell cycle progression and maintenance of genomic stability 11 . Denticleless E3 ubiquitin protein ligase homolog (DTL), also known as CDT2, DCAF2, or RAMP, serves as the substrate receptor of the CRL4 Cdt 2 ubiquitin ligase complex 12 . The unique substrate recognition paradigm of CRL4 Cdt 2 depends on prior interaction of its substrates with chromatin-bound PCNA 13 . DTL is responsible for recognizing multiple substrates such as Cdt1, E2F1, Set8, and p21 13, 14, 15, 16 . To date, the role of CRL4 Cdt 2 in maintaining genome integrity has been well clarified 13 , 17 . Due to the central part of DTL in genome stability and its multiple substrates, misregulation of DTL is associated with tumorigenesis 18 , 19 . Previous studies have shown that overexpression of DTL correlated with adverse outcomes in certain tumors, such as cervical cancer, gastric cancer, and melanoma 20 , 21 , 22 . However, the underlying molecular mechanism for DTL misregulation and the role of DTL in HCC malignant progression remain unclear. In this study, the biological significance of DTL was verified based on a retrospective patient cohort, HCC RNA-sequencing (RNA-seq) data, and public databases (The Cancer Genome Atlas, TCGA and Gene Expression Omnibus, GEO). Functionally, DTL promoted HCC proliferation, metastasis, and sorafenib resistance. Mechanistically, we demonstrated hypoxia-induced DTL expression through a HIF-1α-dependent manner for the first time. Elevated DTL expression activated epithelial-mesenchymal transition (EMT) through the Notch pathway. In general, this study suggested DTL as a promising biomarker of HCC malignant progression. It revealed a novel HIF-1α/DTL/Notch signaling pathway that may serve as a potential target for HCC therapy. Results DTL is upregulated in HCC tissues and associated with poor prognosis RNA Sequencing was performed on matched adjacent nontumor-tumor- portal vein tumor thrombosis (PVTT) tissue pairs from three patients. The results indicated that DTL expression levels increased successively in adjacent nontumor, HCC, and PVTT tissue (Fig. 1 A, B and Supplementary Fig. 1B-D). To further verify this finding, datasets GSE112790, GSE25097, and GSE102079 were retrieved from the GEO database. After normalizing the expression profile, up-regulated expression of DTL was observed in HCC tissues compared with adjacent normal liver tissues in the three datasets (Fig. 1 C and Supplementary Fig. 1A). Similarly, the expression level of DTL in the HCC dataset from the TCGA database was also higher than in normal liver tissue (unpaired or paired) (Fig. 1 D and Supplementary Fig. 2A); HCC patients with higher DTL expression level correlated with shorter overall survival (OS) based on TCGA database (Supplementary Fig. 2B). In addition, DTL was higher in advanced pathological grade, T and N stages (Supplementary Fig. 2C, D and E). The area under the curve (AUC) under the receiver operating characteristics (ROC) curve was 0.926, indicating that DTL had excellent predictive ability for HCC (Supplementary Fig. 2F). Therefore, DTL was selected for further experiments. To explore whether DTL expression was associated with HCC patients’ clinical features and prognosis, we retrospectively collected clinical information and tissue samples after obtaining approval from the Ethics Committee of Sun Yat-sen University Cancer Center. A total of 209 patients who underwent surgical resection and were pathologically confirmed as HCC from January 2017 to January 2019 were included in this study. Immunohistochemistry (IHC) staining was performed on paraffin-embedded tissue sections. DTL expression level in HCC tissue was significantly higher than in adjacent nontumor tissue (P < 0.001, Fig. 1 E and F). According to the cutoff values determined by the X-tile, patients were divided into two groups: DTL high expression (IHC ≥ 6, n = 40) and DTL low expression group (IHC < 6, n = 169). Supplementary table 1 shows the baseline characteristics of patients in both groups. The upregulated expression of DTL was significantly associated with vascular invasion (P < 0.001) and advanced TNM stage (P < 0.001) (Supplementary Table. 1). The results of KM survival analysis suggest that patients with high expression of DTL exhibited a poorer OS (P ≤ 0.001) or disease-free survival (DFS) (P ≤ 0.001) (Fig. 1 G and H). Moreover, the univariate analysis showed that tumor differentiation, tumor size, vascular invasion, TNM stage, and DTL level were significantly associated with OS and DFS in HCC patients, while age stage was associated only with OS (P 3.0 cm) and high DTL level were independent risk factors for both OS and DFS (P < 0.05) (Supplementary Tables 2 and 3). These results indicated that up-regulation of DTL is significantly related to HCC progression and poor clinical prognosis. DTL is induced by hypoxia To find the underlying mechanism of the upregulated expression of DTL in HCC, the TCGA-liver cancer database was used to explore the correlation between DTL and multiple pathways with Spearman correlation analysis. The results indicated that DTL was closely related to the hypoxia-related pathway (Fig. 2 A) 23 , DNA damage repair-related pathways (DNA-repair), and proliferation-related pathways (proliferation-signature, G2M-checkpoint, DNA-replication) (Supplementary Fig. 3). Previous studies have suggested that DTL induces proliferation, metastasis, and invasion of cancer cells through several different signaling pathways 19 , 22 , 24 . As a common feature of solid tumors, the hypoxia microenvironment mediates expression changes in many downstream genes 6 . Therefore, we investigated whether the hypoxia tumor microenvironment is involved in the up-regulated expression of DTL. IHC staining and scoring of HIF-1α and DTL were performed in 209 HCC samples (Fig. 2 B). We found that DTL expression, as expected, positively correlated with HIF-1α(R 2 = 0.409, P < 0.001) (Fig. 2 C). Then, Huh-7 and HCCLM-3 cells were cultured in hypoxic atmosphere (1% O 2 ). qRT-PCR and western blotting were used to detect the expression of HIF-1α and DTL at time points 0, 1, 2, 4, 6, and 8 h. The results showed that DTL mRNA/protein levels increase in response to hypoxia (Fig. 2 D and E). The results described above indicated that abnormal expression of DTL may be caused by hypoxia. HIF-1α activates DTL expression by binding to HRE in the DTL promoter region HIF-1α, a key transcription factor in the microenvironment, induces the expression of its downstream target genes and promotes HCC proliferation, angiogenesis, and metastasis 6 , 8 , 25 . Therefore, we hypothesize that HIF-1α transcriptionally upregulates the expression of DTL under hypoxia conditions. After inhibiting HIF-1α with small interfering RNA (siRNA), the expression of HIF-1α and DTL significantly decreased at both mRNA and protein levels (P < 0.01) (Fig. 3 A, B and Supplementary Fig. 4A). The small-molecule inhibitor KC7F2A, which selectively inhibits HIF-1α mRNA translation, resulted in a significant decrease of HIF-1α and DTL at the protein level (Fig. 3 B). Dual-luciferase reporter assay employed under HIF-1α overexpression or hypoxia conditions, demonstrating that HIF-1α binds to the DTL promoter and activates its transcription (P < 0.001); the use of KC7F2A significantly inhibited luciferase intensity indicating that DTL expression is mainly regulated by HIF-1α under hypoxic conditions (Fig. 3 C). JASPAR transcription factor database was used to detect potential hypoxia response element (HRE) within the − 2000 to + 50 bp region of DTL transcription start site (TSS). We observed two putative HRE within the genomic region (Fig. 3 D). Then, chromatin immunoprecipitation (CHIP) using an antibody to HIF-1α and qRT-PCR was performed on the putative HRE under hypoxia (1% O 2 ) or cobalt chloride (Cocl 2 , 200µM) conditions. These results suggest that HIF-1α could directly bind to both HRE1 and HRE2 of the DTL promoter region in Huh-7 and HCCLM-3 cells (Fig. 3 E and F). Next, we constructed an HRE mutated vector in turn, as shown in Fig. 3 G. Luciferase activity was measured under the conditions of hypoxia, hypoxia + siHIF-1α, and hypoxia + KC7F2; mutating HRE1 or HRE2 respectively resulted in a significant decrease in fluorescence intensity under hypoxic conditions. Additionally, HRE (1 + 2) mutation restored luciferase activity under hypoxic to the normoxic conditions level (Fig. 3 H). Similarly, siHIF-1α or KC7F2 can significantly reduce the luciferase activity of HEK-293T cells under hypoxic conditions, and the luciferase activity of HRE1 or HRE2 mutant group decreased significantly. Moreover, the luciferase activity of HRE1 + 2 mutant group showed no significant changes after siHIF-1α or KC7F2 treatment (Fig. 3 I and J). In light of these results, we can conclude that DTL is a direct transcriptional target of HIF-1α, and HRE1 and 2 are crucial in this process. DTL promotes HCC proliferation and sorafenib resistance To investigate the oncogenic function of DTL in HCC cells, lentivirus-mediated stable overexpression and knockdown of DTL cell lines were constructed. The DTL knockdown cell lines were constructed with a doxycycline-inducible system since the poor growth status was caused by DTL silence. DTL knockdown cell lines were cultured in DMEM containing 10% tetracycline-free serum (ABWBIO, China). The knockdown effect was satisfactory after 48 h of doxycycline induction (1 µg/mL) (Supplementary Fig. 4B). Stable cell lines were verified through qPCR and WB analysis (Fig. 4 A and B). For all subsequent in vitro experiments, DTL-shRNA cell lines were conducted after 48 h of doxycycline induction (1 µg/mL). Cell cycle analysis by flow cytometry was performed to test changes in the Huh-7 and HCCLM-3 cell lines after DTL overexpression and knockdown. Overexpression of DTL promotes cell-cycle through increasing the G2/M phase population, and knockdown of DTL induces G2/M cell-cycle arrest (Fig. 4 C). Cell proliferation ability was detected by CCK8 proliferation assay and colony formation assay. These results suggested that DTL promoted the proliferation of HCC. In turn, silencing of DTL severely slowed growth rates and impaired the colony formation abilities of HCC cells (Fig. 4 D-G). Sorafenib, a multi-kinase inhibitor, is the standard therapy for advanced HCC with limited survival benefit 26 . Multiple genes involved in the proliferation, migration, or invasion of HCC were related to sorafenib resistance 27 . In this study, we found the knockdown of DTL sensitized Huh-7 and HCCLM-3 cells’ response toward sorafenib treatment (Fig. 4 H). Similarly, the CCK-8 assays also indicated that DTL knockdown combined with sorafenib exhibited a more substantial inhibitory effect on HCC cells (Fig. 4 I). DTL promotes HCC invasion and metastasis in vivo and in vitro To explore the effect of DTL on HCC cells migration and invasion ability, we performed in vitro wound-healing and transwell assays. DTL overexpression significantly enhanced HCC cells migration ability and increased the number of cells migrating/invading through the membrane compared to the empty vector. By contrast, DTL knockdown markedly suppressed the mobility and invasiveness of Huh-7 and HCCLM-3 cells (Fig. 5 A-C, Supplementary Fig. 5A and B). Moreover, to detect the effect of DTL on the proliferation and metastasis of HCC in vivo, we contrasted the tail vein metastasis model and orthotopic HCC implantation model with HCCLM-3 DTL-overexpression, knockdown, and their corresponding control cells; paraffin-embedded tissue sections were HE stained to detect the intrahepatic tumor and lung metastatic. The results showed that the number of metastatic lung nodules in the DTL overexpression group (n = 10) was significantly increased compared to the control group (n = 10); the DTL knockdown group (n = 10) displayed the opposite results (Fig. 5 D and F, Supplementary Fig. 5C). Likewise, in the liver orthotopic transplantation model, DTL promoted tumor growth and metastasis in terms of ratio of liver weight/mice weight and intrahepatic metastatic foci compared with the control group (n = 6); in contrast, knockdown of DTL reduced the liver/mice weight ratio and the number of intrahepatic metastatic nodules (n = 10) (Fig. 5 E, G and H, Supplementary Fig. 5D-F). The number of lung metastatic nodules in the liver orthotopic transplantation model was also counted and reached similar conclusions (Fig. 5 I, Supplementary Fig. 5G, and H). These results suggest that DTL enhances HCC cell proliferation, invasion, and metastasis in vivo and in vitro. DTL plays a crucial role in hypoxia-induced proliferation and metastasis of HCC Hypoxia/HIF-1α promotes proliferation and metastasis of HCC cells 7 , 28 . As hypoxia increases DTL expression in HCC, we examined whether DTL was required in mediating hypoxia-induced proliferation and metastasis of HCC cells. As depicted in Fig. 6 A, the suppression of DTL effectively hindered the hypoxia-induced increase of the G2/M cell-cycle phase in both Huh-7 and HCCLM-3 cells. CCK-8 and colony formation assays showed that silencing DTL suppressed the hypoxia-induced proliferation of HCC cells (Fig. 6 B and C). The scratch test and transwell assays indicated that hypoxia-induced cell migration and invasion were attenuated by DTL knockdown (Fig. 6 D and E). All the above results indicate that DTL plays a vital role in hypoxia-induced proliferation and metastasis of HCC. DTL induces EMT through the Notch pathway To elucidate the potential molecular mechanisms underlying the DTL in promoting HCC malignant progression, we performed RNA-seq on DTL overexpression HCCLM-3 cells and its control cell line (Fig. 7 A). Gene set enrichment analysis (GSEA) results suggest that upregulated DTL may activat the Notch signaling pathway (Fig. 7 B). Consistently, the results of qRT-PCR, western blot, and immunofluorescence showed that upregulation of DTL resulted in a significant increase of the Notch1 and EMT related gene levels, while silencing of DTL had an opposite effect (Fig. 7 C-E and Supplementary Fig. 6A). Next, to investigate the impact of Notch pathway on DTL’s oncogenic role in HCC cells, we performed rescue assays with Notch1 small interfering RNA (siNotch1). Both targets on Notch1 siRNA could effectively inhibit the expression of Notch1 at the mRNA and protein level (Fig. 7 F and G, Supplementary Fig. 6B). As shown in Fig. 7 H, I and Supplementary Fig. 6C, the CCK-8 and colony formation assays demonstrated that siNotch1 significantly inhibited DTL-induced proliferation in HCC cells. On the other hand, results of the scratch test and transwell assays suggested that the migration and invasion capabilities induced by DTL were partly reversed by the siNotch1 (Fig. 7 J and K, Supplementary Fig. 6D and E). Taken together, our results suggest that DTL accelerates HCC cells proliferation and movement by activating the Notch signaling pathway. Discussion DTL, a PCNA-dependent E3 ubiquitin ligase, plays a central role in the maintenance of genome integrity. Molecular mechanisms of DTL participating in the physiological cell and DNA damage response have been extensively studied 18 . Given the central role of DTL, recent studies have demonstrated that DTL is highly expressed in various cancers and involved in cancer progression 19 , 22 , 29 . Previous Studies have reported that DTL functionally promotes the cell cycle and affects immune cell infiltration in HCC 30 , 31 . However, the role of DTL in other malignant phenotypes and its underlying molecular mechanisms remain poorly understood. On the other hand, the mechanism triggering the up-regulation of DTL remains unclear. First, to verify the correlation between DTL level and patient prognosis, we recruited the largest HCC cohort to date; data from RNA-seq and public databases were collected and analyzed. The results indicated a significant correlation between high DTL expression and poor prognosis in HCC patients. Furthermore, DTL level showed a significant correlation between higher TNM staging, the absence of vascular invasion, and PVTT, as shown in supplementary table 1 . Subsequently, the functional role of DTL in promoting proliferation and metastasis of HCC was verified through in vivo and in vitro experiments. Due to the absence of vascular invasion or distant metastasis, more than 50% of patients are diagnosed at an advanced stage, and approximately 70% of patients suffer from relapse within 5 years 32 , 33 . Sorafenib is known as a cornerstone treatment for advanced HCC. However, only about 30% of patients are benefitted from sorafenib, and drug resistance usually develops within 6 months 27 . Enhanced proliferation and cell motility are essential for the development of sorafenib resistance. Many genes that participate in the proliferation or migration of HCC cells can promote resistance to sorafenib 34 , 35 . Our results indicated that DTL downregulation improves the sensitivity of HCC cells to sorafenib, which could provide new therapeutic targets for overcoming the drug resistance of sorafenib. As one of the most striking features of solid tumors, the hypoxia tumor microenvironment contributes to the malignant phenotype and aggressive tumor behaviors 36 . HIF-1α is one of the most commonly oncogenic transcriptional regulators and participates in almost all cellular processes 37 . Up-regulation of HIF-1α was reported to promote growth, metastasis, and sorafenib resistance 38 , 39 . While inhibition of HIF-1α has long been considered a potential therapeutic target, effective drugs, as well as deeper mechanisms, still need to be investigated 40 . In our study, we revealed a hypoxia-dependent transcriptional activation molecular mechanism of DTL mediated by HIF-1α for the first time. CHIP assays showed that HIF-1α directly binds to the hypoxia response element (HRE) on the DTL promoter. The up-regulation of DTL under hypoxia was mainly mediated by HIF-1α. Since, in the absence of HRE on the DTL promoter, hypoxia could not induce transcriptional activation. Furthermore, the functional importance of DTL in the hypoxia-induced malignant progression of HCC was also confirmed. These results suggest that DTL is a novel functional mediator downstream of hypoxia/ HIF-1α signaling. Epithelial-mesenchymal transition (EMT) is the initial process of tumor cells taken to invade and metastasize 41 . Under hypoxia, multiple signaling pathways were activated, including TGF-β, NF-κB, phosphoinositide 3-kinase (PI3K)/Akt, and Notch-1 42, 43 . As a highly conserved pathway, the notch signaling pathway plays a critical role in the initiation and progression of different cancers 44 . Notch receptor is a heterodimer receptor composed of the Notch extracellular domain, transmembrane, and Notch intracellular domains (NICD). Once interacting with a transmembrane ligand, the NICD is released and translocates into the nucleus. Then, a ternary complex, formed by NICD and the members of the CSL transcription factor family, mediates the transcription of downstream genes 43 . Pathway analysis of the RNA-seq analysis revealed that DTL expression was positively correlated with the Notch signaling pathway. The expression detection of NICD and several downstream markers indicated that DTL can activate the Notch pathway and facilitate EMT. The DTL-mediated proliferation, metastasis, and EMT of HCC can also be reversed by the siNotch1. The study by Jin et al. indicated that DTL exerted carcinogenic effects by inducing the c-Jun N-terminal kinase (JNK) pathway in cervical adenocarcinoma 22 . Our sequencing results showed that DTL resulted in a more obvious activation of the Notch pathway, which indicated the underlying oncogenic mechanisms of DTL were not the same. However, the mechanism underlying the interaction between DTL and the Notch signaling pathway remains unclear. In summary, our study elucidated that increased DTL expression in HCC was closely related to adverse clinicopathological features and poor prognosis. DTL functions as an oncogene promoting the proliferation, metastasis, and sorafenib resistance of HCC cells. Mechanistically, HIF-1α activates DTL transcription by binding to HRE under hypoxia. DTL exerts its oncogenic by activating the Notch pathway. The functional role of DTL and Notch1 in the HIF-1α/DTL/Notch1 axis was confirmed by suppressing them, respectively. Therefore, we identified a promising therapeutic target for HCC intervention. Further research is necessary to determine the interaction between DTL and the Notch pathway. Materials and Methods Data collection and tissue specimens In this study, three pairs of adjacent non-tumor, HCC, and PVTT tissue were collected with written informed consent and subjected to RNA-Seq analysis. RNA-seq library preparation and sequencing were performed by GENEWIZ (Genewiz, Suzhou, China). Data on HCC gene expression and clinical features were retrospectively collected from TCGA (424 samples) data set ( http://cancergenome.nih.gov/ ) and GEO database ( www.ncbi.nlm.nih.gov/geo ), including GSE 102079, GSE 112790, GSE 25097. Primary HCC tissue and corresponding non-tumor tissue were collected from 209 patients who underwent hepatectomy at the Sun Yat-sen University Cancer Center (Guangzhou, China) from May 2017 to May 2018. Surgical specimens were formalin-fixed and embedded in paraffin for IHC. All included patients were pathologically diagnosed as HCC with complete clinical information and follow-up data. The study protocol was authorized by the Ethics Committee of Sun Yat-sen University Cancer Center (G2022-068-01), and written informed consent was obtained from all participants. Clinical information of the HCC cohort is shown in Supplementary Table 1. Cells and chemicals Huh-7, HCCLM-3, and HEK-293T cells were preserved by Sun Yat-Sen University Cancer Center (Guangzhou, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Waltham, MA, USA) with 10% fetal bovine serum (FBS) (1050S, ABWBIO, Shanghai, China), and 1% penicillin-streptomycin (Gibco). Mycoplasma testing was performed every 3 months to ensure no mycoplasma contamination. All cells were incubated under 5% CO2 at 37 ℃ with 21% O 2, while hypoxia experiments were performed under 1% O 2 conditions. Cocl 2 (60818, Sigma-Aldrich, Saint Louis, MO, USA) was diluted into a 150 mM stock solution with sterile water and added to the medium at the working concentration of 150 µM. KC7F2 (S7946, Selleck Chemicals, Shanghai, China) and Sorafenib (S7397, Selleck Chemicals) were dissolved in dimethyl sulfoxide (DMSO) to 10 mM stock solution. Tetracycline hydrochloride (T105493, Aladdin, Shanghai, China) was used at the working concentration of 1µg/mL (in vitro) and 1mg/mL (in vivo). RNA interference, plasmid constructions, and viral infections siRNAs targeting HIF-1α and Notch1 were provided by Ribo-Bio (Guangzhou, China). The overexpression vectors were constructed by cloning wide type (WT) HIF-1α and DTL cDNA sequences into pCDNA3.1 and pCDH-CMV-MCS-EF1-puro vectors, respectively. Lentiviral vectors expressing tetracycline-inducible shRNA against DTL were purchased from Youming-Bio (Guangzhou, China). The WT DTL promoter was amplified and cloned into PGL3-BASIC vector. The HRE deletion mutation vectors were generated by PCR based on the WT Vector. siRNA or plasmid was transfected into cells using lipofectamine 3000 (Invitrogen, Carlsbad, CA). The Lentivirus containing DTL overexpression or silencing plasmids was transfected into Huh-7 and HCCLM-3 cells. After 24 h, a puromycin-containing medium was used to screen for 2 weeks to obtain stable cell lines. Primer information and target sequences are listed in supplementary tables 4 and 5. Immunohistochemistry and scoring The paraffin-embedded tissue sections were dewaxed with xylene and rehydrated with graded ethanol. The sections were soaked in sodium citrate buffer for antigen retrieval and incubated with 10% goat serum to block the non-specific binding sites. Subsequently, samples were incubated with primary antibodies against HIF-1α (1:200, Cell Signaling Technology, Boston), DTL (1:200; Abcam, Cambridge, UK), and N-cadherin (1:400, Proteintech, Wuhan, China) overnight at 4℃, followed by incubation with corresponding HRP secondary antibodies for 1 h at room temperature. IHC scoring was independently assessed by two pathologists in a double-blinded manner. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from cells using an RNA Purification Kit (EZBioscience, Shanghai, China). Reverse Transcription Kit (EZBioscience) was used to obtain cDNA; qRT-PCR was performed by 2×Color STBR Green qPCR Master Mix (EZBioscience) using CFX96/384 qPCR System (Biorad, Hercules, CA). All primers used for qRT-PCR are listed in Supplementary Table 6. Immunofluorescence assay and western blots assay Immunofluorescence staining was performed as described previously 45 . Briefly, cells grown on coverslips were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.1% Triton X-100 for 10 min. After 1 h of blocking in 5% normal gout serum, coverslips were incubated with primary antibodies at 4℃ overnight. Fluorochrome-conjugated secondary antibodies were incubated for 1 h at room temperature; nuclei were stained with DAPI. Images were captured on a NIKON Eclipse Ti2-U. For western blots, total proteins extracted from cells were separated by 10% Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked by 5% non-fat milk for 1 h and then incubated overnight with primary antibodies at 4℃. After washing, HRP-conjugated secondary antibodies were added and incubated for 40 min. Chemiluminescent signals were developed using Clarity Western ECL substrate (Bio-Rad). Details of primary and secondary antibodies used for immunofluorescence and western blot are listed in Supplementary Table 7. Dual luciferase reporter assay Luciferase reporter plasmid of the DTL promotor and its mutant form were constructed as previously described. HEK-293T cells were seeded in 48-well plates and transfected with constructed firefly luciferase reporter plasmids (100ng) and Renilla luciferase reporter plasmid (40ng). Forty-eight hours after transfection, cells were lysed and luciferase activity was measured by a Dual-Luciferase Reporter Gene Assay Kit (Yeasen Biotechnology, Shanghai, China). Chromatin immunoprecipitation (ChIP) To perform ChIP assays, an EZ-ChIP™ Kit (Merk Millipore, Billerica, MA) was used following the manufacturer’s instructions. Briefly, 3 × 10 7 cells in each immunoprecipitation reaction were crosslinked with 1% formaldehyde, and 0.125 M glycine was added to terminate the reaction. Chromatin DNA was sonicated on ice to obtain 200–1000 bp fragments. The cell lysate was incubated overnight with HIF-1α or IgG antibodies and Protein A/G magnetic beads. After elution and decrosslinking, co-precipitated DNA was quantified by qRT-PCR. Cell cycle analysis All harvested cells were washed twice with ice-cold phosphate-buffered saline, fixed, and permeabilized with 70% ethanol at -20℃ overnight. Subsequently, samples were stained with propidium iodide (50µg/mL) and RNase A (1mg/mL) at room temperature for 30 min. Cell cycle distribution was analyzed using Propidium iodide (PI) staining by flow cytometry (Beckman CytoFlex). Cell counting Kit-8 (CCK-8) assay The HCC cells were seeded into a 96-well plate at the density of 1000/well. Five multiple wells were set up for each group. CCK-8 (Vazyme, Nanjing, China) solution (10µL of reagent in 100µL culture medium) was added into each well and incubated for 2 h. The absorbance value was measured at 450 nm. Clone formation assay HCC cells (1 × 10 3 per well) were seeded in 6-well plates and cultured in a complete culture medium for 2 weeks. The colonies were fixed with methanol and stained with 0.1% crystal violet solution. Later, the number of colonies was counted using Image J software (version 6.0). Wound-healing assay HCC cells were plated at a density of 1 x 10 6 per well in a 6-well plate. When the cell confluency reached 90%, a 200-µL pipette tip was used to scratch the cell monolayer. After washing with PBS, cells were incubated in a serum-free medium. The scratch closure was observed by an inverted microscope (Nikon Eclipse Ti2), and the scratch area was calculated with the Image J software (version 6.0). Transwell assays Before seeding, cells were starved for 24h, and the lower chamber was coated with fibronectin (20µg/mL) for 2 h. The matrigel (Corning, Shenzhen, China) was precoated for the invasion assay in the upper chamber. Then, cells were seeded into the upper chamber with 200µL serum-free medium, and the bottom chambers were filled with 800µL complete medium containing 10-% FBS as a chemoattractant. After culturing for 24h, the cells in the upper chamber were removed. Migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. Cells on the bottom of the membrane were imaged and counted (five random fields per group). Tumor xenograft models For the pulmonary metastasis model, 4-week-old male nude BALB/c mice were randomly assigned to each group. 2 × 10 6 cells suspended in 200µL PBS were injected into the lateral tail veins of mice. Mice were sacrificed 8 weeks later, and the lung tissue was collected, photographed, and fixed in 4% paraformaldehyde. For the orthotopic HCC implantation model, approximately 2 x 10 6 HCC cells were suspended in a 40µL DMEM-Matrigel mixture at a 1:1 ratio. Through a 1cm midline incision in the upper abdomen under anesthesia, 6-week-old male nude BALA/c mice were orthotopically inoculated in the left hepatic lobe with a microsyringe. Six weeks later, all mice were sacrificed, and their livers and lungs were dissected, weighed, and fixed in 4% paraformaldehyde. Hematoxylin-eosin (HE) staining was used to evaluate intrahepatic and lung metastasis. In addition, all mice in DTL knockdown and the control groups were fed with 1 mg/L tetracycline in the drinking water after inoculation of tumor cells to induce DTL knockdown in vivo. All animal studies were approved by the Sun Yat-sen University Cancer Center Animal Experimental Ethics Committee (L1020220210031). Statistical analysis All statistical analysis and graphing were performed using the open-source R statistical software package (version 3.6.3) or GraphPad Prism (version 6.0). Data were shown as mean ± S.E.M. Chi-square test or Student’s t-test was used to compare the difference between the two groups. For multiple comparisons, one-way ANOVA with Tukey’s multiple comparisons test was used. Survival analysis was performed using Kaplan–Meier survival analysis. The Cox proportional hazards regression model was conducted for univariate and multivariate analysis. All analysis were two-tailed, with P < 0.05 considered significant: *P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant. Declarations Acknowledgments We are grateful to Drs. Chao-nan Qian (Department of Nasopharyngeal Carcinoma, Sun Yat-Sen University Cancer Center), Fu-jun Zhang (Department of Imaging and Interventional Radiology, Sun Yat-sen University Cancer Center)forsharing the plasmids and cell lines. We appreciate the encouragement and helpful comments from the Qian laboratory. Conflicts of Interest Statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Authorship Contribution Statement All authors contributed to and approved the final version of this manuscript. F. G. and B.J. H. performed study concept and design; Z.X. C. performed the experiments and drafted the original manuscript; The collection of tissue specimens and clinical information as well as data analysis were performed by M.Y. M. and G. Y.; H. Q., X.B. F., G.S. W., and W.W. J.: review and revision of the paper. Ethics Statement The study protocol was authorized by the Ethics Committee of Sun Yat-sen University Cancer Center (Guangzhou, China), and written informed consent was obtained from all participants. Funding Statement This study was supported by grants from the Natural Science Foundation of Guangdong Province(2022A1515010490) and Beijing Xisike Clinical Oncology Research Foundation (Y-bayer202002-0089). Data Availability Statement The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin 2023, 73(1): 17–48. Vogel A, Meyer T, Sapisochin G, Salem R, Saborowski A. Hepatocellular carcinoma. Lancet 2022, 400(10360): 1345–1362. Yang C, Zhang H, Zhang L, Zhu AX, Bernards R, Qin W, et al. Evolving therapeutic landscape of advanced hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 2023, 20(4): 203–222. Zucman-Rossi J, Villanueva A, Nault JC, Llovet JM. Genetic Landscape and Biomarkers of Hepatocellular Carcinoma. Gastroenterology 2015, 149(5): 1226–1239 e1224. Corzo CA, Condamine T, Lu L, Cotter MJ, Youn JI, Cheng P, et al. HIF-1α regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment. The Journal of experimental medicine 2010, 207(11): 2439–2453. Sin SQ, Mohan CD, Goh RMW, You M, Nayak SC, Chen L, et al. Hypoxia signaling in hepatocellular carcinoma: Challenges and therapeutic opportunities. Cancer Metastasis Rev 2022. Wu XZ, Xie GR, Chen D. Hypoxia and hepatocellular carcinoma: The therapeutic target for hepatocellular carcinoma. Journal of gastroenterology and hepatology 2007, 22(8): 1178–1182. Yuen VW, Wong CC. Hypoxia-inducible factors and innate immunity in liver cancer. J Clin Invest 2020, 130(10): 5052–5062. Yamashita K, Discher DJ, Hu J, Bishopric NH, Webster KA. Molecular regulation of the endothelin-1 gene by hypoxia. Contributions of hypoxia-inducible factor-1, activator protein-1, GATA-2, AND p300/CBP. The Journal of biological chemistry 2001, 276(16): 12645–12653. Befani C, Liakos P. The role of hypoxia-inducible factor-2 alpha in angiogenesis. Journal of cellular physiology 2018, 233(12): 9087–9098. Jang SM, Redon CE, Aladjem MI. Chromatin-Bound Cullin-Ring Ligases: Regulatory Roles in DNA Replication and Potential Targeting for Cancer Therapy. Frontiers in molecular biosciences 2018, 5: 19. Panagopoulos A, Taraviras S, Nishitani H, Lygerou Z. CRL4(Cdt2): Coupling Genome Stability to Ubiquitination. Trends Cell Biol 2020, 30(4): 290–302. Havens CG, Walter JC. Mechanism of CRL4(Cdt2), a PCNA-dependent E3 ubiquitin ligase. Genes Dev 2011, 25(15): 1568–1582. Pozo PN, Cook JG. Regulation and Function of Cdt1; A Key Factor in Cell Proliferation and Genome Stability. Genes 2016, 8(1). Shibutani ST, de la Cruz AF, Tran V, Turbyfill WJ, 3rd, Reis T, Edgar BA, et al. Intrinsic negative cell cycle regulation provided by PIP box- and Cul4Cdt2-mediated destruction of E2f1 during S phase. Developmental cell 2008, 15(6): 890–900. Galanos P, Vougas K, Walter D, Polyzos A, Maya-Mendoza A, Haagensen EJ, et al. Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing. Nature cell biology 2016, 18(7): 777–789. Abbas T, Dutta A. CRL4Cdt2: master coordinator of cell cycle progression and genome stability. Cell Cycle 2011, 10(2): 241–249. Jin J, Arias EE, Chen J, Harper JW, Walter JC. A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. Mol Cell 2006, 23(5): 709–721. Cui H, Wang Q, Lei Z, Feng M, Zhao Z, Wang Y, et al. DTL promotes cancer progression by PDCD4 ubiquitin-dependent degradation. J Exp Clin Cancer Res 2019, 38(1): 350. Kobayashi H, Komatsu S, Ichikawa D, Kawaguchi T, Hirajima S, Miyamae M, et al. Overexpression of denticleless E3 ubiquitin protein ligase homolog (DTL) is related to poor outcome in gastric carcinoma. Oncotarget 2015, 6(34): 36615–36624. Benamar M, Guessous F, Du K, Corbett P, Obeid J, Gioeli D, et al. Inactivation of the CRL4-CDT2-SET8/p21 ubiquitylation and degradation axis underlies the therapeutic efficacy of pevonedistat in melanoma. EBioMedicine 2016, 10: 85–100. Liu S, Gu L, Wu N, Song J, Yan J, Yang S, et al. Overexpression of DTL enhances cell motility and promotes tumor metastasis in cervical adenocarcinoma by inducing RAC1-JNK-FOXO1 axis. Cell Death Dis 2021, 12(10): 929. Cockman ME, Masson N, Mole DR, Jaakkola P, Chang GW, Clifford SC, et al. Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein. The Journal of biological chemistry 2000, 275(33): 25733–25741. Chen YC, Chen IS, Huang GJ, Kang CH, Wang KC, Tsao MJ, et al. Targeting DTL induces cell cycle arrest and senescence and suppresses cell growth and colony formation through TPX2 inhibition in human hepatocellular carcinoma cells. Onco Targets Ther 2018, 11: 1601–1616. Wicks EE, Semenza GL. Hypoxia-inducible factors: cancer progression and clinical translation. J Clin Invest 2022, 132(11). Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. The New England journal of medicine 2008, 359(4): 378–390. Tang W, Chen Z, Zhang W, Cheng Y, Zhang B, Wu F, et al. The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects. Signal Transduct Target Ther 2020, 5(1): 87. Gwak GY, Yoon JH, Kim KM, Lee HS, Chung JW, Gores GJ. Hypoxia stimulates proliferation of human hepatoma cells through the induction of hexokinase II expression. J Hepatol 2005, 42(3): 358–364. Feng M, Wang Y, Bi L, Zhang P, Wang H, Zhao Z, et al. CRL4A(DTL) degrades DNA-PKcs to modulate NHEJ repair and induce genomic instability and subsequent malignant transformation. Oncogene 2021, 40(11): 2096–2111. Li Z, Wang R, Qiu C, Cao C, Zhang J, Ge J, et al. Role of DTL in Hepatocellular Carcinoma and Its Impact on the Tumor Microenvironment. Front Immunol 2022, 13: 834606. Pan HW, Chou HY, Liu SH, Peng SY, Liu CL, Hsu HC. Role of L2DTL, cell cycle-regulated nuclear and centrosome protein, in aggressive hepatocellular carcinoma. Cell Cycle 2006, 5(22): 2676–2687. Reig M, Forner A, Rimola J, Ferrer-Fabrega J, Burrel M, Garcia-Criado A, et al. BCLC strategy for prognosis prediction and treatment recommendation: The 2022 update. J Hepatol 2022, 76(3): 681–693. Llovet JM, Zucman-Rossi J, Pikarsky E, Sangro B, Schwartz M, Sherman M, et al. Hepatocellular carcinoma. Nature reviews Disease primers 2016, 2: 16018. Quagliata L, Quintavalle C, Lanzafame M, Matter MS, Novello C, di Tommaso L, et al. High expression of HOXA13 correlates with poorly differentiated hepatocellular carcinomas and modulates sorafenib response in in vitro models. Laboratory investigation; a journal of technical methods and pathology 2018, 98(1): 95–105. Xia P, Zhang H, Xu K, Jiang X, Gao M, Wang G, et al. MYC-targeted WDR4 promotes proliferation, metastasis, and sorafenib resistance by inducing CCNB1 translation in hepatocellular carcinoma. Cell Death Dis 2021, 12(7): 691. L.Harris A. HYPOXIA — A KEY REGULATORY FACTOR IN TUMOUR GROWTH. Nat Rev Cancer 2002 Jan, 2(1):38–47. Ma Z, Wang LZ, Cheng JT, Lam WST, Ma X, Xiang X, et al. Targeting Hypoxia-Inducible Factor-1-Mediated Metastasis for Cancer Therapy. Antioxidants & redox signaling 2021, 34(18): 1484–1497. Dou C, Zhou Z, Xu Q, Liu Z, Zeng Y, Wang Y, et al. Hypoxia-induced TUFT1 promotes the growth and metastasis of hepatocellular carcinoma by activating the Ca(2+)/PI3K/AKT pathway. Oncogene 2019, 38(8): 1239–1255. Song Z, Liu T, Chen J, Ge C, Zhao F, Zhu M, et al. HIF-1alpha-induced RIT1 promotes liver cancer growth and metastasis and its deficiency increases sensitivity to sorafenib. Cancer Lett 2019, 460: 96–107. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003, 3(10): 721–732. Yang MH, Mohan CD, Deivasigamani A, Chinnathambi A, Alharbi SA, Rangappa KS, et al. Procaine Abrogates the Epithelial-Mesenchymal Transition Process through Modulating c-Met Phosphorylation in Hepatocellular Carcinoma. Cancers 2022, 14(20). Gurzu S, Kobori L, Fodor D, Jung I. Epithelial Mesenchymal and Endothelial Mesenchymal Transitions in Hepatocellular Carcinoma: A Review. BioMed research international 2019, 2019: 2962580. Guo M, Niu Y, Xie M, Liu X, Li X. Notch signaling, hypoxia, and cancer. Frontiers in Oncology 2023, 13. Artavanis-Tsakonas S, Muskavitch MA. Notch: the past, the present, and the future. Current topics in developmental biology 2010, 92: 1–29. Cao L, Qi L, Zhang L, Song W, Yu Y, Xu C, et al. Human nonsense-mediated RNA decay regulates EMT by targeting the TGF-ß signaling pathway in lung adenocarcinoma. Cancer Lett 2017, 403: 246–259. Mackintosh C, Ordóñez JL, García-Domínguez DJ, Sevillano V, Llombart-Bosch A, Szuhai K, et al. 1q gain and CDT2 overexpression underlie an aggressive and highly proliferative form of Ewing sarcoma. Oncogene 2012, 31(10): 1287–1298. Additional Declarations (Not answered) Supplementary Files SupplementaryFigure1.tif Supplementary Figure 1: (A) Heatmap of DEGs for GSE102079, GSE112790, and GSE25097 datasets from GEO, respectively. (B-D). Volcano plot of hepatocellular carcinoma (HCC) vs. adjacent nontumor, portal vein tumor thrombosis (PVTT) vs. adjacent nontumor, and PVTT vs HCC, respectively. SupplementaryFigure2.tif Supplementary Figure 2: (A) DTL mRNA levels between HCC samples and matched liver tissue (n=50) from the TCGA database. (B) Kaplan-Meier survival analysis of OS for HCC patients with high and low expression of DTL from TCGA database. (C-E) DTL mRNA levels among different histological grade (C), pathologic T stage (D), and pathologic N stage (E) in the TCGA-LIHC cohort. (F) The capacity to identify tumor or normal tissue sources of DTL in the TCGA-LIHC cohort. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance SupplementaryFigure3.tif Supplementary Figure 3: Spearman correlation analysis between DTL and pathway score. 46 The signal pathway positively correlated with the overexpression of DTL. SupplementaryFigure4.tif Supplementary Figure 4: (A) The mRNA level of HIF-1α and DTL was determined after HIF-1α-siRNA transfection or KC7F2 (40 μM) treatment under hypoxia (1% O2) in HCCLM-3 cells. (B) The effectiveness of doxycycline-induced DTL knockdown in Huh-7 and HCCLM-3 cells was detected in various concentrations. Knockdown of DTL was mediated by treatment with 1.0 μg/mL doxycycline for 48 h in further experiments. SupplementaryFigure5.tif Supplementary Figure 5: (A) Transwell assays were performed to evaluate the invasion ability of DTL overexpression and knockdown HCC cells. (B) Representative images of wound healing assay in HCC cells. (C) Representative images of lung tissues in tail vein tumor metastasis mouse model from DTL knockdown and negative control (NC) groups. (D) Representative images of liver tissues in orthotopic liver transplantation model from DTL knockdown and negative control (NC) groups. (E) Liver weight in orthotopic liver transplantation model. (F) Representative images of intrahepatic metastasis nodules in HE‐stained sections. Left panel: the number of intrahepatic metastasis nodules in the DTL overexpression group. (G and H) Representative images of lung tissues in orthotopic liver transplantation model from DTL overexpression (G) and knockdown (H) groups. SupplementaryFigure6.tif Supplementary Figure 6: (A) The immunofluorescence results of Notch1 and N-cadherin expression in DTL overexpressing or knockdown Huh-7 cells. (B) The knockdown efficiency of Notch1 by siRNA was detected by qRT-PCR in HCCLM-3 cells. (C) The functional role of Notch1 in the DTL-induced proliferation of HCCLM-3 cells was detected by Cell Counting Kit-8 (CCK8) (D and E). The functional role of Notch1 in DTL-induced metastasis of HCCLM-3 cells was detected by wound-healing and transwell assays. SupplementaryTable.docx Supplementary Table 1-7 Fulluneditedblotsforfigure4B.tif Full unedited blots for figure 4B FullUneditedBlotsforFigure2Eand3B.tif Fulluneditedblotsforfigure4B.tif Fulluneditedblotsforfigure7G.tif Fulluneditedblotsforfigure7D.tif Full unedited blots for figure 7D SupplementaryTable.docx ajchecklist.pdf Cite Share Download PDF Status: Published Journal Publication published 09 Oct, 2024 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 15 Apr, 2024 Review # 4 received at journal 28 Mar, 2024 Review # 3 received at journal 10 Mar, 2024 Reviewer # 4 agreed at journal 09 Mar, 2024 Reviewer # 3 agreed at journal 19 Feb, 2024 Review # 2 received at journal 12 Jan, 2024 Reviewer # 2 agreed at journal 04 Jan, 2024 Reviewer # 1 agreed at journal 04 Jan, 2024 Reviewers invited by journal 04 Jan, 2024 Submission checks completed at journal 01 Dec, 2023 First submitted to journal 01 Dec, 2023 Editor assigned by journal 01 Dec, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3691309","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":265165642,"identity":"2976c302-cd7b-4419-84ab-7ba956abb331","order_by":0,"name":"Fei Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYJCCDx9I1cE4cwbJWmbzkKRe3v3wwWabP4ftDY4ffsDwcU8tA//sBvxaDM+kJTbnth1O3HAmzYBxxrPjDBJ3DhDQ0pBj/ji34XCCwQ0eBmaeA8cYDCQSCGjpf2PYbAFyGNFa5CVyDJsZ2A4zboBoqSGsxUDiWWJjb1t64kygXw7OOHCAR+IGIVv6kw82/Phjbc93/PDDBx8O1MnxzyBkywEw1QwmgezDhONIvgFM1cH4dbgUjoJRMApGwQgGAKtLSAXlfAyMAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4086-2575","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":true,"prefix":"","firstName":"Fei","middleName":"","lastName":"Gao","suffix":""},{"id":265165643,"identity":"9ec21bc9-451d-47cd-a520-dd066f89ad7f","order_by":1,"name":"Zi-Xiong Chen","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Zi-Xiong","middleName":"","lastName":"Chen","suffix":""},{"id":265165644,"identity":"431fdb2b-acef-4f80-8007-ff6ad2af73cc","order_by":2,"name":"Mao-Yuan Mu","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Mao-Yuan","middleName":"","lastName":"Mu","suffix":""},{"id":265165645,"identity":"b7fd7ee4-3582-4410-bc8a-d86902053a5a","order_by":3,"name":"Guang Yang","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Guang","middleName":"","lastName":"Yang","suffix":""},{"id":265165646,"identity":"74fd895d-d1cc-44c3-8ce0-8d996c918ad6","order_by":4,"name":"Han Qi","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Qi","suffix":""},{"id":265165647,"identity":"6f1800e0-f438-47d0-9f7b-b425854c6db7","order_by":5,"name":"Xiao-Bo Fu","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Xiao-Bo","middleName":"","lastName":"Fu","suffix":""},{"id":265165648,"identity":"1d802cf4-d8be-47d5-93fa-6766c3c3ee6c","order_by":6,"name":"Gui-Song Wang","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Gui-Song","middleName":"","lastName":"Wang","suffix":""},{"id":265165649,"identity":"533adf3f-3cf3-495e-8856-2d553db0cb14","order_by":7,"name":"Wei-Wei Jiang","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Wei-Wei","middleName":"","lastName":"Jiang","suffix":""},{"id":265165650,"identity":"bc2dffa6-addf-400b-beb7-4f29de98390f","order_by":8,"name":"Bi-Jun Huang","email":"","orcid":"https://orcid.org/0000-0001-6816-1503","institution":"State Key Laboratory of Oncology in South China","correspondingAuthor":false,"prefix":"","firstName":"Bi-Jun","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2023-12-01 08:51:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3691309/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3691309/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-024-07089-4","type":"published","date":"2024-10-09T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49331192,"identity":"96862486-8e50-416c-953e-39af162f301b","added_by":"auto","created_at":"2024-01-08 19:15:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1444331,"visible":true,"origin":"","legend":"\u003cp\u003eDTL is overexpressed in human HCCs and correlates with poor prognosis. (A) Heatmap of mRNA sequencing data from three pairs of PVTT, tumor and adjacent nontumor tissues. Venn diagrams displayed the overlap DEGs among (B) three paired samples and (C) three HCC data sets. (D) DTL transcriptional expression in patients with HCC and adjacent nontumor from profiling data on TCGA databases. (E) Representative IHC staining showed the DTL expression level in HCC and adjacent nontumor tissues (×100). (F) IHC score analysis of DTL in HCC and adjacent nontumor samples (n=209). (G and H) Disease-free survival and overall survival in DTL-high and DTL-low groups. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns, no significance\u003c/p\u003e","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/e95f85c7b987b12dbd7d53c4.png"},{"id":49331193,"identity":"7467316e-9886-4de8-ad8e-93c03b831c18","added_by":"auto","created_at":"2024-01-08 19:15:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2541534,"visible":true,"origin":"","legend":"\u003cp\u003eDTL expression was positively correlated with hypoxia. (A) The correlation between DTL and cellular response to the hypoxia pathway was analyzed with Spearman. (B) Representative IHC staining of HIF-1α, DTL, and N-cadherin in DTL-high and DTL-low HCC tissue. (C) Correlation analysis between the IHC score of DTL and HIF-1α (n=209). (D) The mRNA levels of DTL at different hypoxia time points. (E) Protein expression of DTL and HIF-1α at different time points. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns, no significance\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/79544aee540dc8246fc82a67.png"},{"id":49331982,"identity":"b011d843-d8c2-40e0-9eae-fbf0564fe5f2","added_by":"auto","created_at":"2024-01-08 19:23:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1208036,"visible":true,"origin":"","legend":"\u003cp\u003eHIF-1α activates DTL transcription under hypoxia. (A and B) HCC cells were transfected with HIF-1α-siRNA or treated with KC7F2 (40 μM) under hypoxia (1% O2). The mRNA and protein expression of DTL and HIF-1α were determined using qRT-PCR and a western blot assay. (C) Dual luciferase reporter assays were performed in HEK-293T cells with HIF-1α overexpression or hypoxia following KC7F2 treatment (40 μM). (D) Sequences of the predicted HRE in the DTL promoter. (E and F) Chromatin immunoprecipitation (ChIP) assays in Huh-7 and HCCLM-3 cells to identify the binding of HIF-1α to the DTL promoter under hypoxia or after treatment with Cocl2. (G) Schematic diagram of the DTL promoter and three mutant constructs. (H) Dual luciferase reporter assays for the mutant HRE sequences in HEK-293T cells under hypoxia. (I and J) Dual luciferase reporter assays for the mutant HRE sequences in HEK-293T cells following siHIF-1α and KC7F2 (40 μM) under hypoxia. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns, no significance\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/85a4a66873d70b845b2f6d58.png"},{"id":49331196,"identity":"c653beec-c62a-4d2f-88a0-ac3dc19f7437","added_by":"auto","created_at":"2024-01-08 19:15:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2068609,"visible":true,"origin":"","legend":"\u003cp\u003eDTL promotes cell cycle progression, proliferation, and sorafenib resistance in HCC cells. (A and B) DTL overexpression and knockdown stable cell line were established; the DTL mRNA and protein levels were determined by qRT-PCR and western blot. (C) The effect of DTL overexpression and knockdown on the cell cycle in Huh-7 and HCCLM-3 were analyzed by flow cytometry. (D and E) The effect of DTL overexpression and knockdown on proliferation in Huh-7 and HCCLM-3 cells was determined by Cell Counting Kit-8 (CCK8) assays. (F and G) The effect of DTL overexpression and knockdown on proliferation in Huh-7 and HCCLM-3 cells was determined by clone formation assay. (H) The relative cell viability of DTL knockdown Huh-7 and HCCLM-3 cells after treatment with different concentrations of sorafenib for 72 h was detected by CCK-8 assays. (I) CCK-8 assays showed the proliferation of Huh-7 and HCCLM-3 cells response to 3μM sorafenib with DTL knockdown. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns, no significance\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/3157bdbcd5782f41f1c9be7f.png"},{"id":49331194,"identity":"bc1263c2-a56b-4672-8863-d585c0be4a71","added_by":"auto","created_at":"2024-01-08 19:15:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4443863,"visible":true,"origin":"","legend":"\u003cp\u003eDTL promoted metastasis of HCC cells in vitro and vivo. (A and B) The effect of DTL overexpression and knockdown on Huh-7 and HCCLM-3 cells wound healing. (C) Transwell assays were performed to evaluate the migration ability of DTL overexpression and knockdown HCC cells. (D) Representative images of lung tissues in tail vein tumor metastasis mouse model from DTL overexpression and vector groups. (E) Representative images of liver tissues in orthotopic liver transplantation model from DTL overexpression and vector groups. (F) representative images of lung metastatic nodules in hematoxylin-eosin (HE) stained sections. The left panel shown the number of lung nodules in DTL overexpression and vector groups from the tail vein tumor metastasis mouse model. (G) The relative liver weight was calculated as the ratio of wet liver weight to body weight ratio. (H) Representative images of intrahepatic metastasis nodules in HE‐stained sections. Left panel: the number of intrahepatic metastasis nodules in the DTL knockdown group. (I) Representative images of lung metastatic nodules in HE‐stained sections. Left panel: the number of lung nodules in DTL overexpression and vector groups from orthotopic liver transplantation model. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns, no significance\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/40dc4cea910f39f6d0bcabfa.png"},{"id":49331197,"identity":"4321a76e-ee72-4e1c-a77b-32a98c6a0312","added_by":"auto","created_at":"2024-01-08 19:15:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4597271,"visible":true,"origin":"","legend":"\u003cp\u003eThe oncogenic role of HIF-1α in HCC cells partly depended on DTL. (A) Cell cycle analysis evaluated the effects of DTL knockdown on cell cycle under hypoxia in HCC cells. (B and C) Cell Counting Kit-8 (CCK8) and clone formation assays were performed to evaluate the DTL knockdown on the proliferation of HCC cells under hypoxia. (D) Representative images and quantification of wound-healing assays for DTL knockdown HCC cells under hypoxia. (E) Representative images and quantification of transwell migration and invasion assays for DTL knockdown HCC cells under hypoxia. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns, no significance\u003c/p\u003e","description":"","filename":"OnlineFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/aefb32a5b5d75fb4af50fa68.png"},{"id":49331201,"identity":"bc8909e4-9d39-4697-ab7a-b415a7024aaf","added_by":"auto","created_at":"2024-01-08 19:16:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4486967,"visible":true,"origin":"","legend":"\u003cp\u003eNotch signaling pathway mediated DTL-induced EMT, proliferation, and metastasis of HCC cells. (A) Heatmap of the representative up-regulated and down-regulated gene after overexpression of DTL in HCCLM-3 cells. (B) Gene Set Enrichment Analysis (GSEA) of the Notch pathway. (C) qRT-PCR results of Notch1 and EMT-related genes mRNA expression in DTL overexpressing or knockdown HCC cells. (D) Western blot results of Notch1 and EMT-related genes protein expression in DTL overexpressing or knockdown HCC cells. (E) Immunofluorescence results of Notch1 and N-cadherin expression in DTL overexpressing or knockdown HCCLM-3 cells. (F and G) qRT-PCR and western blot detected the knockdown efficiency of Notch1 by siRNA. (H and I) The functional role of Notch1 in DTL-induced proliferation of HCC cells was detected by Cell Counting Kit-8 (CCK8) and clone formation assays. (J and K) The functional role of Notch1 in DTL-induced metastasis of Huh-7 cells was detected by wound-healing and transwell assays. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns, no significance\u003c/p\u003e","description":"","filename":"OnlineFig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/631c89701c0ea15e32cfb64d.png"},{"id":66304059,"identity":"70510db6-c349-44b7-867b-3c5174e57c8b","added_by":"auto","created_at":"2024-10-10 07:06:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4082841,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/68e7c1e8-099f-4f59-9af5-f643c799759d.pdf"},{"id":49331212,"identity":"aef17898-5999-4635-a1db-2a8de7445eb4","added_by":"auto","created_at":"2024-01-08 19:16:01","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19509748,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 1: (A) Heatmap of DEGs for GSE102079, GSE112790, and GSE25097 datasets from GEO, respectively. (B-D). Volcano plot of hepatocellular carcinoma (HCC) vs. adjacent nontumor, portal vein tumor thrombosis (PVTT) vs. adjacent nontumor, and PVTT vs HCC, respectively.\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/13387c68c8b23f6c97bdae6c.tif"},{"id":49331205,"identity":"2855fe49-b312-4503-a187-da8202c05b3a","added_by":"auto","created_at":"2024-01-08 19:16:00","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15037976,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 2: (A) DTL mRNA levels between HCC samples and matched liver tissue (n=50) from the TCGA database. (B) Kaplan-Meier survival analysis of OS for HCC patients with high and low expression of DTL from TCGA database. (C-E) DTL mRNA levels among different histological grade (C), pathologic T stage (D), and pathologic N stage (E) in the TCGA-LIHC cohort. (F) The capacity to identify tumor or normal tissue sources of DTL in the TCGA-LIHC cohort. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns, no significance\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/f5e86ffdb855e14f090e6231.tif"},{"id":49331198,"identity":"86530f57-72ed-47df-afce-a4093072c180","added_by":"auto","created_at":"2024-01-08 19:15:59","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11525792,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 3: Spearman correlation analysis between DTL and pathway score. \u003csup\u003e46\u003c/sup\u003e The signal pathway positively correlated with the overexpression of DTL.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"SupplementaryFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/106dda466a80421bbb5509c8.tif"},{"id":49331199,"identity":"68fe8d7a-6c4c-4d4a-9b73-9e54a61e7fcd","added_by":"auto","created_at":"2024-01-08 19:15:59","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11975944,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 4: (A) The mRNA level of HIF-1α and DTL was determined after HIF-1α-siRNA transfection or KC7F2 (40 μM) treatment under hypoxia (1% O2) in HCCLM-3 cells. (B) The effectiveness of doxycycline-induced DTL knockdown in Huh-7 and HCCLM-3 cells was detected in various concentrations. Knockdown of DTL was mediated by treatment with 1.0 μg/mL doxycycline for 48 h in further experiments.\u003c/p\u003e","description":"","filename":"SupplementaryFigure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/c8ee02c0ec1525cfe74771e2.tif"},{"id":49331210,"identity":"a4a48f04-e709-435d-94f0-406a3eae9ea7","added_by":"auto","created_at":"2024-01-08 19:16:00","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":27939800,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 5: (A) Transwell assays were performed to evaluate the invasion ability of DTL overexpression and knockdown HCC cells. (B) Representative images of wound healing assay in HCC cells. (C) Representative images of lung tissues in tail vein tumor metastasis mouse model from DTL knockdown and negative control (NC) groups. (D) Representative images of liver tissues in orthotopic liver transplantation model from DTL knockdown and negative control (NC) groups. (E) Liver weight in orthotopic liver transplantation model. (F) Representative images of intrahepatic metastasis nodules in HE‐stained sections. Left panel: the number of intrahepatic metastasis nodules in the DTL overexpression group. (G and H) Representative images of lung tissues in orthotopic liver transplantation model from DTL overexpression (G) and knockdown (H) groups.\u003c/p\u003e","description":"","filename":"SupplementaryFigure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/a62f49d35c11d610111fb3d5.tif"},{"id":49331211,"identity":"381ab736-10b6-4723-afa4-0299313bc9a1","added_by":"auto","created_at":"2024-01-08 19:16:00","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":19820784,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 6: (A) The immunofluorescence results of Notch1 and N-cadherin expression in DTL overexpressing or knockdown Huh-7 cells. (B) The knockdown efficiency of Notch1 by siRNA was detected by qRT-PCR in HCCLM-3 cells. (C) The functional role of Notch1 in the DTL-induced proliferation of HCCLM-3 cells was detected by Cell Counting Kit-8 (CCK8) (D and E). The functional role of Notch1 in DTL-induced metastasis of HCCLM-3 cells was detected by wound-healing and transwell assays.\u003c/p\u003e","description":"","filename":"SupplementaryFigure6.tif","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/0bdc5c595a1eda056e799f87.tif"},{"id":49331202,"identity":"b25ad70d-a48a-4e1d-b6d2-dd79ad46ee12","added_by":"auto","created_at":"2024-01-08 19:16:00","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":31593,"visible":true,"origin":"","legend":"Supplementary Table 1-7","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/5f8850894748592215f5eb3c.docx"},{"id":49331207,"identity":"1bbb44a8-7816-4798-adfb-5354335a3ee5","added_by":"auto","created_at":"2024-01-08 19:16:00","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":27687960,"visible":true,"origin":"","legend":"Full unedited blots for figure 4B","description":"","filename":"Fulluneditedblotsforfigure4B.tif","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/e636e46afb30c53f379448d3.tif"},{"id":49331215,"identity":"3396285a-5a67-44fc-8fca-9ecc63e1f924","added_by":"auto","created_at":"2024-01-08 19:16:02","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":29819424,"visible":true,"origin":"","legend":"","description":"","filename":"FullUneditedBlotsforFigure2Eand3B.tif","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/5817b697c2514347c8551a73.tif"},{"id":49331213,"identity":"f5acd938-697e-47e7-b775-e9d2c04c00af","added_by":"auto","created_at":"2024-01-08 19:16:01","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":27687960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fulluneditedblotsforfigure4B.tif","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/4050203526ed464afd60f96b.tif"},{"id":49331985,"identity":"49eca766-ca4c-435f-9084-aeaaba81ab68","added_by":"auto","created_at":"2024-01-08 19:24:01","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":24571560,"visible":true,"origin":"","legend":"","description":"","filename":"Fulluneditedblotsforfigure7G.tif","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/85f369dfd1e9294ed0594746.tif"},{"id":49331209,"identity":"382a589f-4b5e-4b68-8d35-6e8df5f80cf1","added_by":"auto","created_at":"2024-01-08 19:16:00","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":38802520,"visible":true,"origin":"","legend":"Full unedited blots for figure 7D","description":"","filename":"Fulluneditedblotsforfigure7D.tif","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/13a66c5e5acf96d232912bbc.tif"},{"id":49331208,"identity":"6bc7cb33-f1ff-43c6-b99e-3df5f42be6d4","added_by":"auto","created_at":"2024-01-08 19:16:00","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":31593,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/b574179b96ab76b4b57ba3b5.docx"},{"id":49331983,"identity":"8d97c9cf-00c2-4e51-be1a-79f92fca6bda","added_by":"auto","created_at":"2024-01-08 19:24:00","extension":"pdf","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":1908300,"visible":true,"origin":"","legend":"","description":"","filename":"ajchecklist.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3691309/v1/d1d4e91276390e94fce5fe9c.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Hypoxia-induced DTL promotes the proliferation, metastasis, and sorafenib resistance of Hepatocellular Carcinoma through Notch Signaling Pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWith the fastest-increasing mortality for decades, liver cancer ranks as the third largest cancer-related death worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Hepatocellular carcinoma (HCC) accounts for ~\u0026thinsp;90% of primary liver cancer, with a 5-year relative survival rate of approximately 18%\u003csup\u003e2\u003c/sup\u003e. Moreover, two-thirds of HCC patients are diagnosed at advanced stages with limited treatment options and worse prognosis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Whilst our understanding of the molecular pathogenesis of HCC has been improved, few effective molecular targets or biomarkers have been translated into clinical practice\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Therefore, there is an urgent need to further understand the mechanisms underlying the HCC progression.\u003c/p\u003e \u003cp\u003eHypoxia, as the primary characteristic of the tumor microenvironment, plays a vital role in the progression of HCC\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. As a hypermetabolic tumor, the uninhibited proliferation and abnormal formation of micro-vessels make HCC more susceptible to hypoxia\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Hypoxia-inducible transcription factor 1α (HIF-1α), the primary regulator of cellular adaptive responses to hypoxia, extensively participates in the malignant progression and resistance to chemotherapy and immunotherapy of HCC\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In the absence of oxygen, HIF-1α binds to hypoxia-responsive elements (HREs) and enhances transcription of downstream target genes\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The representative cancer-related genes regulated by HIF-1α include the vascular endothelial growth factor (VEGF), transforming growth factor (TGF-β), and P53\u003csup\u003e10\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases, are essential for many eukaryotic biological processes, including cell cycle progression and maintenance of genomic stability\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Denticleless E3 ubiquitin protein ligase homolog (DTL), also known as CDT2, DCAF2, or RAMP, serves as the substrate receptor of the CRL4\u003csup\u003eCdt\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e ubiquitin ligase complex\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The unique substrate recognition paradigm of CRL4\u003csup\u003eCdt\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e depends on prior interaction of its substrates with chromatin-bound PCNA\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. DTL is responsible for recognizing multiple substrates such as Cdt1, E2F1, Set8, and p21\u003csup\u003e13, 14, 15, 16\u003c/sup\u003e. To date, the role of CRL4\u003csup\u003eCdt\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e in maintaining genome integrity has been well clarified\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Due to the central part of DTL in genome stability and its multiple substrates, misregulation of DTL is associated with tumorigenesis\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that overexpression of DTL correlated with adverse outcomes in certain tumors, such as cervical cancer, gastric cancer, and melanoma\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, the underlying molecular mechanism for DTL misregulation and the role of DTL in HCC malignant progression remain unclear.\u003c/p\u003e \u003cp\u003eIn this study, the biological significance of DTL was verified based on a retrospective patient cohort, HCC RNA-sequencing (RNA-seq) data, and public databases (The Cancer Genome Atlas, TCGA and Gene Expression Omnibus, GEO). Functionally, DTL promoted HCC proliferation, metastasis, and sorafenib resistance. Mechanistically, we demonstrated hypoxia-induced DTL expression through a HIF-1α-dependent manner for the first time. Elevated DTL expression activated epithelial-mesenchymal transition (EMT) through the Notch pathway. In general, this study suggested DTL as a promising biomarker of HCC malignant progression. It revealed a novel HIF-1α/DTL/Notch signaling pathway that may serve as a potential target for HCC therapy.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDTL is upregulated in HCC tissues and associated with poor prognosis\u003c/h2\u003e \u003cp\u003eRNA Sequencing was performed on matched adjacent nontumor-tumor- portal vein tumor thrombosis (PVTT) tissue pairs from three patients. The results indicated that DTL expression levels increased successively in adjacent nontumor, HCC, and PVTT tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B and Supplementary Fig.\u0026nbsp;1B-D). To further verify this finding, datasets GSE112790, GSE25097, and GSE102079 were retrieved from the GEO database. After normalizing the expression profile, up-regulated expression of DTL was observed in HCC tissues compared with adjacent normal liver tissues in the three datasets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Supplementary Fig.\u0026nbsp;1A). Similarly, the expression level of DTL in the HCC dataset from the TCGA database was also higher than in normal liver tissue (unpaired or paired) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Supplementary Fig.\u0026nbsp;2A); HCC patients with higher DTL expression level correlated with shorter overall survival (OS) based on TCGA database (Supplementary Fig.\u0026nbsp;2B). In addition, DTL was higher in advanced pathological grade, T and N stages (Supplementary Fig.\u0026nbsp;2C, D and E). The area under the curve (AUC) under the receiver operating characteristics (ROC) curve was 0.926, indicating that DTL had excellent predictive ability for HCC (Supplementary Fig.\u0026nbsp;2F). Therefore, DTL was selected for further experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore whether DTL expression was associated with HCC patients\u0026rsquo; clinical features and prognosis, we retrospectively collected clinical information and tissue samples after obtaining approval from the Ethics Committee of Sun Yat-sen University Cancer Center. A total of 209 patients who underwent surgical resection and were pathologically confirmed as HCC from January 2017 to January 2019 were included in this study. Immunohistochemistry (IHC) staining was performed on paraffin-embedded tissue sections. DTL expression level in HCC tissue was significantly higher than in adjacent nontumor tissue (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F).\u003c/p\u003e \u003cp\u003eAccording to the cutoff values determined by the X-tile, patients were divided into two groups: DTL high expression (IHC\u0026thinsp;\u0026ge;\u0026thinsp;6, n\u0026thinsp;=\u0026thinsp;40) and DTL low expression group (IHC\u0026thinsp;\u0026lt;\u0026thinsp;6, n\u0026thinsp;=\u0026thinsp;169). Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the baseline characteristics of patients in both groups. The upregulated expression of DTL was significantly associated with vascular invasion (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and advanced TNM stage (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Supplementary Table. 1). The results of KM survival analysis suggest that patients with high expression of DTL exhibited a poorer OS (P\u0026thinsp;\u0026le;\u0026thinsp;0.001) or disease-free survival (DFS) (P\u0026thinsp;\u0026le;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and H). Moreover, the univariate analysis showed that tumor differentiation, tumor size, vascular invasion, TNM stage, and DTL level were significantly associated with OS and DFS in HCC patients, while age stage was associated only with OS (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); Multivariate analysis demonstrated that tumor size (\u0026gt;\u0026thinsp;3.0 cm) and high DTL level were independent risk factors for both OS and DFS (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Supplementary Tables\u0026nbsp;2 and 3). These results indicated that up-regulation of DTL is significantly related to HCC progression and poor clinical prognosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDTL is induced by hypoxia\u003c/h2\u003e \u003cp\u003eTo find the underlying mechanism of the upregulated expression of DTL in HCC, the TCGA-liver cancer database was used to explore the correlation between DTL and multiple pathways with Spearman correlation analysis. The results indicated that DTL was closely related to the hypoxia-related pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, DNA damage repair-related pathways (DNA-repair), and proliferation-related pathways (proliferation-signature, G2M-checkpoint, DNA-replication) (Supplementary Fig.\u0026nbsp;3). Previous studies have suggested that DTL induces proliferation, metastasis, and invasion of cancer cells through several different signaling pathways\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. As a common feature of solid tumors, the hypoxia microenvironment mediates expression changes in many downstream genes\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Therefore, we investigated whether the hypoxia tumor microenvironment is involved in the up-regulated expression of DTL. IHC staining and scoring of HIF-1α and DTL were performed in 209 HCC samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We found that DTL expression, as expected, positively correlated with HIF-1α(R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.409, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Then, Huh-7 and HCCLM-3 cells were cultured in hypoxic atmosphere (1% O\u003csub\u003e2\u003c/sub\u003e). qRT-PCR and western blotting were used to detect the expression of HIF-1α and DTL at time points 0, 1, 2, 4, 6, and 8 h. The results showed that DTL mRNA/protein levels increase in response to hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and E). The results described above indicated that abnormal expression of DTL may be caused by hypoxia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHIF-1α activates DTL expression by binding to HRE in the DTL promoter region\u003c/h2\u003e \u003cp\u003eHIF-1α, a key transcription factor in the microenvironment, induces the expression of its downstream target genes and promotes HCC proliferation, angiogenesis, and metastasis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Therefore, we hypothesize that HIF-1α transcriptionally upregulates the expression of DTL under hypoxia conditions.\u003c/p\u003e \u003cp\u003eAfter inhibiting HIF-1α with small interfering RNA (siRNA), the expression of HIF-1α and DTL significantly decreased at both mRNA and protein levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B and Supplementary Fig.\u0026nbsp;4A). The small-molecule inhibitor KC7F2A, which selectively inhibits HIF-1α mRNA translation, resulted in a significant decrease of HIF-1α and DTL at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Dual-luciferase reporter assay employed under HIF-1α overexpression or hypoxia conditions, demonstrating that HIF-1α binds to the DTL promoter and activates its transcription (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001); the use of KC7F2A significantly inhibited luciferase intensity indicating that DTL expression is mainly regulated by HIF-1α under hypoxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). JASPAR transcription factor database was used to detect potential hypoxia response element (HRE) within the \u0026minus;\u0026thinsp;2000 to +\u0026thinsp;50 bp region of DTL transcription start site (TSS). We observed two putative HRE within the genomic region (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Then, chromatin immunoprecipitation (CHIP) using an antibody to HIF-1α and qRT-PCR was performed on the putative HRE under hypoxia (1% O\u003csub\u003e2\u003c/sub\u003e) or cobalt chloride (Cocl\u003csub\u003e2\u003c/sub\u003e, 200\u0026micro;M) conditions. These results suggest that HIF-1α could directly bind to both HRE1 and HRE2 of the DTL promoter region in Huh-7 and HCCLM-3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). Next, we constructed an HRE mutated vector in turn, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG. Luciferase activity was measured under the conditions of hypoxia, hypoxia\u0026thinsp;+\u0026thinsp;siHIF-1α, and hypoxia\u0026thinsp;+\u0026thinsp;KC7F2; mutating HRE1 or HRE2 respectively resulted in a significant decrease in fluorescence intensity under hypoxic conditions. Additionally, HRE (1\u0026thinsp;+\u0026thinsp;2) mutation restored luciferase activity under hypoxic to the normoxic conditions level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Similarly, siHIF-1α or KC7F2 can significantly reduce the luciferase activity of HEK-293T cells under hypoxic conditions, and the luciferase activity of HRE1 or HRE2 mutant group decreased significantly. Moreover, the luciferase activity of HRE1\u0026thinsp;+\u0026thinsp;2 mutant group showed no significant changes after siHIF-1α or KC7F2 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI and J). In light of these results, we can conclude that DTL is a direct transcriptional target of HIF-1α, and HRE1 and 2 are crucial in this process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDTL promotes HCC proliferation and sorafenib resistance\u003c/h2\u003e \u003cp\u003eTo investigate the oncogenic function of DTL in HCC cells, lentivirus-mediated stable overexpression and knockdown of DTL cell lines were constructed. The DTL knockdown cell lines were constructed with a doxycycline-inducible system since the poor growth status was caused by DTL silence. DTL knockdown cell lines were cultured in DMEM containing 10% tetracycline-free serum (ABWBIO, China). The knockdown effect was satisfactory after 48 h of doxycycline induction (1 \u0026micro;g/mL) (Supplementary Fig.\u0026nbsp;4B). Stable cell lines were verified through qPCR and WB analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). For all subsequent in vitro experiments, DTL-shRNA cell lines were conducted after 48 h of doxycycline induction (1 \u0026micro;g/mL). Cell cycle analysis by flow cytometry was performed to test changes in the Huh-7 and HCCLM-3 cell lines after DTL overexpression and knockdown. Overexpression of DTL promotes cell-cycle through increasing the G2/M phase population, and knockdown of DTL induces G2/M cell-cycle arrest (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Cell proliferation ability was detected by CCK8 proliferation assay and colony formation assay. These results suggested that DTL promoted the proliferation of HCC. In turn, silencing of DTL severely slowed growth rates and impaired the colony formation abilities of HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSorafenib, a multi-kinase inhibitor, is the standard therapy for advanced HCC with limited survival benefit\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Multiple genes involved in the proliferation, migration, or invasion of HCC were related to sorafenib resistance\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In this study, we found the knockdown of DTL sensitized Huh-7 and HCCLM-3 cells\u0026rsquo; response toward sorafenib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Similarly, the CCK-8 assays also indicated that DTL knockdown combined with sorafenib exhibited a more substantial inhibitory effect on HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDTL promotes HCC invasion and metastasis in vivo and in vitro\u003c/h2\u003e \u003cp\u003eTo explore the effect of DTL on HCC cells migration and invasion ability, we performed in vitro wound-healing and transwell assays. DTL overexpression significantly enhanced HCC cells migration ability and increased the number of cells migrating/invading through the membrane compared to the empty vector. By contrast, DTL knockdown markedly suppressed the mobility and invasiveness of Huh-7 and HCCLM-3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C, Supplementary Fig.\u0026nbsp;5A and B). Moreover, to detect the effect of DTL on the proliferation and metastasis of HCC in vivo, we contrasted the tail vein metastasis model and orthotopic HCC implantation model with HCCLM-3 DTL-overexpression, knockdown, and their corresponding control cells; paraffin-embedded tissue sections were HE stained to detect the intrahepatic tumor and lung metastatic. The results showed that the number of metastatic lung nodules in the DTL overexpression group (n\u0026thinsp;=\u0026thinsp;10) was significantly increased compared to the control group (n\u0026thinsp;=\u0026thinsp;10); the DTL knockdown group (n\u0026thinsp;=\u0026thinsp;10) displayed the opposite results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and F, Supplementary Fig.\u0026nbsp;5C). Likewise, in the liver orthotopic transplantation model, DTL promoted tumor growth and metastasis in terms of ratio of liver weight/mice weight and intrahepatic metastatic foci compared with the control group (n\u0026thinsp;=\u0026thinsp;6); in contrast, knockdown of DTL reduced the liver/mice weight ratio and the number of intrahepatic metastatic nodules (n\u0026thinsp;=\u0026thinsp;10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, G and H, Supplementary Fig.\u0026nbsp;5D-F). The number of lung metastatic nodules in the liver orthotopic transplantation model was also counted and reached similar conclusions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, Supplementary Fig.\u0026nbsp;5G, and H). These results suggest that DTL enhances HCC cell proliferation, invasion, and metastasis in vivo and in vitro.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDTL plays a crucial role in hypoxia-induced proliferation and metastasis of HCC\u003c/h2\u003e \u003cp\u003eHypoxia/HIF-1α promotes proliferation and metastasis of HCC cells\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. As hypoxia increases DTL expression in HCC, we examined whether DTL was required in mediating hypoxia-induced proliferation and metastasis of HCC cells. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the suppression of DTL effectively hindered the hypoxia-induced increase of the G2/M cell-cycle phase in both Huh-7 and HCCLM-3 cells. CCK-8 and colony formation assays showed that silencing DTL suppressed the hypoxia-induced proliferation of HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and C). The scratch test and transwell assays indicated that hypoxia-induced cell migration and invasion were attenuated by DTL knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and E). All the above results indicate that DTL plays a vital role in hypoxia-induced proliferation and metastasis of HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDTL induces EMT through the Notch pathway\u003c/h2\u003e \u003cp\u003eTo elucidate the potential molecular mechanisms underlying the DTL in promoting HCC malignant progression, we performed RNA-seq on DTL overexpression HCCLM-3 cells and its control cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Gene set enrichment analysis (GSEA) results suggest that upregulated DTL may activat the Notch signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Consistently, the results of qRT-PCR, western blot, and immunofluorescence showed that upregulation of DTL resulted in a significant increase of the Notch1 and EMT related gene levels, while silencing of DTL had an opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-E and Supplementary Fig.\u0026nbsp;6A). Next, to investigate the impact of Notch pathway on DTL\u0026rsquo;s oncogenic role in HCC cells, we performed rescue assays with Notch1 small interfering RNA (siNotch1). Both targets on Notch1 siRNA could effectively inhibit the expression of Notch1 at the mRNA and protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF and G, Supplementary Fig.\u0026nbsp;6B). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, I and Supplementary Fig.\u0026nbsp;6C, the CCK-8 and colony formation assays demonstrated that siNotch1 significantly inhibited DTL-induced proliferation in HCC cells. On the other hand, results of the scratch test and transwell assays suggested that the migration and invasion capabilities induced by DTL were partly reversed by the siNotch1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ and K, Supplementary Fig.\u0026nbsp;6D and E). Taken together, our results suggest that DTL accelerates HCC cells proliferation and movement by activating the Notch signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDTL, a PCNA-dependent E3 ubiquitin ligase, plays a central role in the maintenance of genome integrity. Molecular mechanisms of DTL participating in the physiological cell and DNA damage response have been extensively studied\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Given the central role of DTL, recent studies have demonstrated that DTL is highly expressed in various cancers and involved in cancer progression\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Previous Studies have reported that DTL functionally promotes the cell cycle and affects immune cell infiltration in HCC\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, the role of DTL in other malignant phenotypes and its underlying molecular mechanisms remain poorly understood. On the other hand, the mechanism triggering the up-regulation of DTL remains unclear.\u003c/p\u003e \u003cp\u003eFirst, to verify the correlation between DTL level and patient prognosis, we recruited the largest HCC cohort to date; data from RNA-seq and public databases were collected and analyzed. The results indicated a significant correlation between high DTL expression and poor prognosis in HCC patients. Furthermore, DTL level showed a significant correlation between higher TNM staging, the absence of vascular invasion, and PVTT, as shown in supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Subsequently, the functional role of DTL in promoting proliferation and metastasis of HCC was verified through in vivo and in vitro experiments. Due to the absence of vascular invasion or distant metastasis, more than 50% of patients are diagnosed at an advanced stage, and approximately 70% of patients suffer from relapse within 5 years \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Sorafenib is known as a cornerstone treatment for advanced HCC. However, only about 30% of patients are benefitted from sorafenib, and drug resistance usually develops within 6 months\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Enhanced proliferation and cell motility are essential for the development of sorafenib resistance. Many genes that participate in the proliferation or migration of HCC cells can promote resistance to sorafenib\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Our results indicated that DTL downregulation improves the sensitivity of HCC cells to sorafenib, which could provide new therapeutic targets for overcoming the drug resistance of sorafenib.\u003c/p\u003e \u003cp\u003eAs one of the most striking features of solid tumors, the hypoxia tumor microenvironment contributes to the malignant phenotype and aggressive tumor behaviors\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. HIF-1α is one of the most commonly oncogenic transcriptional regulators and participates in almost all cellular processes\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Up-regulation of HIF-1α was reported to promote growth, metastasis, and sorafenib resistance\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. While inhibition of HIF-1α has long been considered a potential therapeutic target, effective drugs, as well as deeper mechanisms, still need to be investigated\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In our study, we revealed a hypoxia-dependent transcriptional activation molecular mechanism of DTL mediated by HIF-1α for the first time. CHIP assays showed that HIF-1α directly binds to the hypoxia response element (HRE) on the DTL promoter. The up-regulation of DTL under hypoxia was mainly mediated by HIF-1α. Since, in the absence of HRE on the DTL promoter, hypoxia could not induce transcriptional activation. Furthermore, the functional importance of DTL in the hypoxia-induced malignant progression of HCC was also confirmed. These results suggest that DTL is a novel functional mediator downstream of hypoxia/ HIF-1α signaling.\u003c/p\u003e \u003cp\u003eEpithelial-mesenchymal transition (EMT) is the initial process of tumor cells taken to invade and metastasize\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Under hypoxia, multiple signaling pathways were activated, including TGF-β, NF-κB, phosphoinositide 3-kinase (PI3K)/Akt, and Notch-1\u003csup\u003e42, 43\u003c/sup\u003e. As a highly conserved pathway, the notch signaling pathway plays a critical role in the initiation and progression of different cancers\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Notch receptor is a heterodimer receptor composed of the Notch extracellular domain, transmembrane, and Notch intracellular domains (NICD). Once interacting with a transmembrane ligand, the NICD is released and translocates into the nucleus. Then, a ternary complex, formed by NICD and the members of the CSL transcription factor family, mediates the transcription of downstream genes\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Pathway analysis of the RNA-seq analysis revealed that DTL expression was positively correlated with the Notch signaling pathway. The expression detection of NICD and several downstream markers indicated that DTL can activate the Notch pathway and facilitate EMT. The DTL-mediated proliferation, metastasis, and EMT of HCC can also be reversed by the siNotch1. The study by Jin et al. indicated that DTL exerted carcinogenic effects by inducing the c-Jun N-terminal kinase (JNK) pathway in cervical adenocarcinoma\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Our sequencing results showed that DTL resulted in a more obvious activation of the Notch pathway, which indicated the underlying oncogenic mechanisms of DTL were not the same. However, the mechanism underlying the interaction between DTL and the Notch signaling pathway remains unclear.\u003c/p\u003e \u003cp\u003eIn summary, our study elucidated that increased DTL expression in HCC was closely related to adverse clinicopathological features and poor prognosis. DTL functions as an oncogene promoting the proliferation, metastasis, and sorafenib resistance of HCC cells. Mechanistically, HIF-1α activates DTL transcription by binding to HRE under hypoxia. DTL exerts its oncogenic by activating the Notch pathway. The functional role of DTL and Notch1 in the HIF-1α/DTL/Notch1 axis was confirmed by suppressing them, respectively. Therefore, we identified a promising therapeutic target for HCC intervention. Further research is necessary to determine the interaction between DTL and the Notch pathway.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData collection and tissue specimens\u003c/h2\u003e \u003cp\u003eIn this study, three pairs of adjacent non-tumor, HCC, and PVTT tissue were collected with written informed consent and subjected to RNA-Seq analysis. RNA-seq library preparation and sequencing were performed by GENEWIZ (Genewiz, Suzhou, China). Data on HCC gene expression and clinical features were retrospectively collected from TCGA (424 samples) data set (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cancergenome.nih.gov/\u003c/span\u003e\u003cspan address=\"http://cancergenome.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://cancergenome.nih.gov/\" target=\"_blank\"\u003ewww.ncbi.nlm.nih.gov/geo\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/geo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), including GSE 102079, GSE 112790, GSE 25097. Primary HCC tissue and corresponding non-tumor tissue were collected from 209 patients who underwent hepatectomy at the Sun Yat-sen University Cancer Center (Guangzhou, China) from May 2017 to May 2018. Surgical specimens were formalin-fixed and embedded in paraffin for IHC. All included patients were pathologically diagnosed as HCC with complete clinical information and follow-up data. The study protocol was authorized by the Ethics Committee of Sun Yat-sen University Cancer Center (G2022-068-01), and written informed consent was obtained from all participants. Clinical information of the HCC cohort is shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCells and chemicals\u003c/h2\u003e \u003cp\u003eHuh-7, HCCLM-3, and HEK-293T cells were preserved by Sun Yat-Sen University Cancer Center (Guangzhou, China) and cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Gibco, Waltham, MA, USA) with 10% fetal bovine serum (FBS) (1050S, ABWBIO, Shanghai, China), and 1% penicillin-streptomycin (Gibco). Mycoplasma testing was performed every 3 months to ensure no mycoplasma contamination. All cells were incubated under 5% CO2 at 37 ℃ with 21% O\u003csub\u003e2,\u003c/sub\u003e while hypoxia experiments were performed under 1% O\u003csub\u003e2\u003c/sub\u003e conditions. Cocl\u003csub\u003e2\u003c/sub\u003e (60818, Sigma-Aldrich, Saint Louis, MO, USA) was diluted into a 150 mM stock solution with sterile water and added to the medium at the working concentration of 150 \u0026micro;M. KC7F2 (S7946, Selleck Chemicals, Shanghai, China) and Sorafenib (S7397, Selleck Chemicals) were dissolved in dimethyl sulfoxide (DMSO) to 10 mM stock solution. Tetracycline hydrochloride (T105493, Aladdin, Shanghai, China) was used at the working concentration of 1\u0026micro;g/mL (in vitro) and 1mg/mL (in vivo).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA interference, plasmid constructions, and viral infections\u003c/h2\u003e \u003cp\u003esiRNAs targeting HIF-1α and Notch1 were provided by Ribo-Bio (Guangzhou, China). The overexpression vectors were constructed by cloning wide type (WT) HIF-1α and DTL cDNA sequences into pCDNA3.1 and pCDH-CMV-MCS-EF1-puro vectors, respectively. Lentiviral vectors expressing tetracycline-inducible shRNA against DTL were purchased from Youming-Bio (Guangzhou, China). The WT DTL promoter was amplified and cloned into PGL3-BASIC vector. The HRE deletion mutation vectors were generated by PCR based on the WT Vector. siRNA or plasmid was transfected into cells using lipofectamine 3000 (Invitrogen, Carlsbad, CA). The Lentivirus containing DTL overexpression or silencing plasmids was transfected into Huh-7 and HCCLM-3 cells. After 24 h, a puromycin-containing medium was used to screen for 2 weeks to obtain stable cell lines. Primer information and target sequences are listed in supplementary tables 4 and 5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry and scoring\u003c/h2\u003e \u003cp\u003eThe paraffin-embedded tissue sections were dewaxed with xylene and rehydrated with graded ethanol. The sections were soaked in sodium citrate buffer for antigen retrieval and incubated with 10% goat serum to block the non-specific binding sites. Subsequently, samples were incubated with primary antibodies against HIF-1α (1:200, Cell Signaling Technology, Boston), DTL (1:200; Abcam, Cambridge, UK), and N-cadherin (1:400, Proteintech, Wuhan, China) overnight at 4℃, followed by incubation with corresponding HRP secondary antibodies for 1 h at room temperature. IHC scoring was independently assessed by two pathologists in a double-blinded manner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells using an RNA Purification Kit (EZBioscience, Shanghai, China). Reverse Transcription Kit (EZBioscience) was used to obtain cDNA; qRT-PCR was performed by 2\u0026times;Color STBR Green qPCR Master Mix (EZBioscience) using CFX96/384 qPCR System (Biorad, Hercules, CA). All primers used for qRT-PCR are listed in Supplementary Table\u0026nbsp;6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence assay and western blots assay\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Briefly, cells grown on coverslips were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.1% Triton X-100 for 10 min. After 1 h of blocking in 5% normal gout serum, coverslips were incubated with primary antibodies at 4℃ overnight. Fluorochrome-conjugated secondary antibodies were incubated for 1 h at room temperature; nuclei were stained with DAPI. Images were captured on a NIKON Eclipse Ti2-U.\u003c/p\u003e \u003cp\u003eFor western blots, total proteins extracted from cells were separated by 10% Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked by 5% non-fat milk for 1 h and then incubated overnight with primary antibodies at 4℃. After washing, HRP-conjugated secondary antibodies were added and incubated for 40 min. Chemiluminescent signals were developed using Clarity Western ECL substrate (Bio-Rad). Details of primary and secondary antibodies used for immunofluorescence and western blot are listed in Supplementary Table\u0026nbsp;7.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDual luciferase reporter assay\u003c/h2\u003e \u003cp\u003eLuciferase reporter plasmid of the DTL promotor and its mutant form were constructed as previously described. HEK-293T cells were seeded in 48-well plates and transfected with constructed firefly luciferase reporter plasmids (100ng) and Renilla luciferase reporter plasmid (40ng). Forty-eight hours after transfection, cells were lysed and luciferase activity was measured by a Dual-Luciferase Reporter Gene Assay Kit (Yeasen Biotechnology, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP)\u003c/h2\u003e \u003cp\u003eTo perform ChIP assays, an EZ-ChIP\u0026trade; Kit (Merk Millipore, Billerica, MA) was used following the manufacturer\u0026rsquo;s instructions. Briefly, 3 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells in each immunoprecipitation reaction were crosslinked with 1% formaldehyde, and 0.125 M glycine was added to terminate the reaction. Chromatin DNA was sonicated on ice to obtain 200\u0026ndash;1000 bp fragments. The cell lysate was incubated overnight with HIF-1α or IgG antibodies and Protein A/G magnetic beads. After elution and decrosslinking, co-precipitated DNA was quantified by qRT-PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis\u003c/h2\u003e \u003cp\u003eAll harvested cells were washed twice with ice-cold phosphate-buffered saline, fixed, and permeabilized with 70% ethanol at -20℃ overnight. Subsequently, samples were stained with propidium iodide (50\u0026micro;g/mL) and RNase A (1mg/mL) at room temperature for 30 min. Cell cycle distribution was analyzed using Propidium iodide (PI) staining by flow cytometry (Beckman CytoFlex).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCell counting Kit-8 (CCK-8) assay\u003c/h2\u003e \u003cp\u003eThe HCC cells were seeded into a 96-well plate at the density of 1000/well. Five multiple wells were set up for each group. CCK-8 (Vazyme, Nanjing, China) solution (10\u0026micro;L of reagent in 100\u0026micro;L culture medium) was added into each well and incubated for 2 h. The absorbance value was measured at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eClone formation assay\u003c/h2\u003e \u003cp\u003eHCC cells (1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e per well) were seeded in 6-well plates and cultured in a complete culture medium for 2 weeks. The colonies were fixed with methanol and stained with 0.1% crystal violet solution. Later, the number of colonies was counted using Image J software (version 6.0).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eWound-healing assay\u003c/h2\u003e \u003cp\u003eHCC cells were plated at a density of 1 x 10\u003csup\u003e6\u003c/sup\u003e per well in a 6-well plate. When the cell confluency reached 90%, a 200-\u0026micro;L pipette tip was used to scratch the cell monolayer. After washing with PBS, cells were incubated in a serum-free medium. The scratch closure was observed by an inverted microscope (Nikon Eclipse Ti2), and the scratch area was calculated with the Image J software (version 6.0).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eTranswell assays\u003c/h2\u003e \u003cp\u003eBefore seeding, cells were starved for 24h, and the lower chamber was coated with fibronectin (20\u0026micro;g/mL) for 2 h. The matrigel (Corning, Shenzhen, China) was precoated for the invasion assay in the upper chamber. Then, cells were seeded into the upper chamber with 200\u0026micro;L serum-free medium, and the bottom chambers were filled with 800\u0026micro;L complete medium containing 10-% FBS as a chemoattractant. After culturing for 24h, the cells in the upper chamber were removed. Migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. Cells on the bottom of the membrane were imaged and counted (five random fields per group).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eTumor xenograft models\u003c/h2\u003e \u003cp\u003eFor the pulmonary metastasis model, 4-week-old male nude BALB/c mice were randomly assigned to each group. 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells suspended in 200\u0026micro;L PBS were injected into the lateral tail veins of mice. Mice were sacrificed 8 weeks later, and the lung tissue was collected, photographed, and fixed in 4% paraformaldehyde. For the orthotopic HCC implantation model, approximately 2 x 10\u003csup\u003e6\u003c/sup\u003e HCC cells were suspended in a 40\u0026micro;L DMEM-Matrigel mixture at a 1:1 ratio. Through a 1cm midline incision in the upper abdomen under anesthesia, 6-week-old male nude BALA/c mice were orthotopically inoculated in the left hepatic lobe with a microsyringe. Six weeks later, all mice were sacrificed, and their livers and lungs were dissected, weighed, and fixed in 4% paraformaldehyde. Hematoxylin-eosin (HE) staining was used to evaluate intrahepatic and lung metastasis. In addition, all mice in DTL knockdown and the control groups were fed with 1 mg/L tetracycline in the drinking water after inoculation of tumor cells to induce DTL knockdown in vivo. All animal studies were approved by the Sun Yat-sen University Cancer Center Animal Experimental Ethics Committee (L1020220210031).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analysis and graphing were performed using the open-source R statistical software package (version 3.6.3) or GraphPad Prism (version 6.0). Data were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. Chi-square test or Student\u0026rsquo;s t-test was used to compare the difference between the two groups. For multiple comparisons, one-way ANOVA with Tukey\u0026rsquo;s multiple comparisons test was used. Survival analysis was performed using Kaplan\u0026ndash;Meier survival analysis. The Cox proportional hazards regression model was conducted for univariate and multivariate analysis. All analysis were two-tailed, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered significant: *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ns, nonsignificant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Drs. Chao-nan Qian (Department of Nasopharyngeal Carcinoma, Sun Yat-Sen University Cancer Center), Fu-jun Zhang (Department of Imaging and Interventional Radiology, Sun Yat-sen University Cancer Center)forsharing the plasmids and cell lines. We appreciate the encouragement and helpful comments from the Qian laboratory.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to and approved the final version of this manuscript. F. G. and B.J. H. performed study concept and design; Z.X. C. performed the experiments and drafted the original manuscript; The collection of tissue specimens and clinical information as well as data analysis were performed by M.Y. M. and G. Y.; H. Q., X.B. F., G.S. W., and W.W. J.: review and revision of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was authorized by the Ethics Committee of Sun Yat-sen University Cancer Center (Guangzhou, China), and written informed consent was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the Natural Science Foundation of Guangdong Province(2022A1515010490) and Beijing Xisike Clinical Oncology Research Foundation (Y-bayer202002-0089).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin 2023, 73(1): 17\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVogel A, Meyer T, Sapisochin G, Salem R, Saborowski A. Hepatocellular carcinoma. Lancet 2022, 400(10360): 1345\u0026ndash;1362.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang C, Zhang H, Zhang L, Zhu AX, Bernards R, Qin W, \u003cem\u003eet al.\u003c/em\u003e Evolving therapeutic landscape of advanced hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 2023, 20(4): 203\u0026ndash;222.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZucman-Rossi J, Villanueva A, Nault JC, Llovet JM. Genetic Landscape and Biomarkers of Hepatocellular Carcinoma. Gastroenterology 2015, 149(5): 1226\u0026ndash;1239 e1224.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorzo CA, Condamine T, Lu L, Cotter MJ, Youn JI, Cheng P, \u003cem\u003eet al.\u003c/em\u003e HIF-1α regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment. The Journal of experimental medicine 2010, 207(11): 2439\u0026ndash;2453.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSin SQ, Mohan CD, Goh RMW, You M, Nayak SC, Chen L, \u003cem\u003eet al.\u003c/em\u003e Hypoxia signaling in hepatocellular carcinoma: Challenges and therapeutic opportunities. Cancer Metastasis Rev 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu XZ, Xie GR, Chen D. Hypoxia and hepatocellular carcinoma: The therapeutic target for hepatocellular carcinoma. Journal of gastroenterology and hepatology 2007, 22(8): 1178\u0026ndash;1182.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuen VW, Wong CC. Hypoxia-inducible factors and innate immunity in liver cancer. J Clin Invest 2020, 130(10): 5052\u0026ndash;5062.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamashita K, Discher DJ, Hu J, Bishopric NH, Webster KA. Molecular regulation of the endothelin-1 gene by hypoxia. Contributions of hypoxia-inducible factor-1, activator protein-1, GATA-2, AND p300/CBP. The Journal of biological chemistry 2001, 276(16): 12645\u0026ndash;12653.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBefani C, Liakos P. The role of hypoxia-inducible factor-2 alpha in angiogenesis. Journal of cellular physiology 2018, 233(12): 9087\u0026ndash;9098.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJang SM, Redon CE, Aladjem MI. Chromatin-Bound Cullin-Ring Ligases: Regulatory Roles in DNA Replication and Potential Targeting for Cancer Therapy. Frontiers in molecular biosciences 2018, 5: 19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePanagopoulos A, Taraviras S, Nishitani H, Lygerou Z. CRL4(Cdt2): Coupling Genome Stability to Ubiquitination. Trends Cell Biol 2020, 30(4): 290\u0026ndash;302.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHavens CG, Walter JC. Mechanism of CRL4(Cdt2), a PCNA-dependent E3 ubiquitin ligase. Genes Dev 2011, 25(15): 1568\u0026ndash;1582.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePozo PN, Cook JG. Regulation and Function of Cdt1; A Key Factor in Cell Proliferation and Genome Stability. Genes 2016, 8(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShibutani ST, de la Cruz AF, Tran V, Turbyfill WJ, 3rd, Reis T, Edgar BA, \u003cem\u003eet al.\u003c/em\u003e Intrinsic negative cell cycle regulation provided by PIP box- and Cul4Cdt2-mediated destruction of E2f1 during S phase. Developmental cell 2008, 15(6): 890\u0026ndash;900.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalanos P, Vougas K, Walter D, Polyzos A, Maya-Mendoza A, Haagensen EJ, \u003cem\u003eet al.\u003c/em\u003e Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing. Nature cell biology 2016, 18(7): 777\u0026ndash;789.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbbas T, Dutta A. CRL4Cdt2: master coordinator of cell cycle progression and genome stability. Cell Cycle 2011, 10(2): 241\u0026ndash;249.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin J, Arias EE, Chen J, Harper JW, Walter JC. A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. Mol Cell 2006, 23(5): 709\u0026ndash;721.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui H, Wang Q, Lei Z, Feng M, Zhao Z, Wang Y, \u003cem\u003eet al.\u003c/em\u003e DTL promotes cancer progression by PDCD4 ubiquitin-dependent degradation. J Exp Clin Cancer Res 2019, 38(1): 350.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKobayashi H, Komatsu S, Ichikawa D, Kawaguchi T, Hirajima S, Miyamae M, \u003cem\u003eet al.\u003c/em\u003e Overexpression of denticleless E3 ubiquitin protein ligase homolog (DTL) is related to poor outcome in gastric carcinoma. Oncotarget 2015, 6(34): 36615\u0026ndash;36624.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenamar M, Guessous F, Du K, Corbett P, Obeid J, Gioeli D, \u003cem\u003eet al.\u003c/em\u003e Inactivation of the CRL4-CDT2-SET8/p21 ubiquitylation and degradation axis underlies the therapeutic efficacy of pevonedistat in melanoma. EBioMedicine 2016, 10: 85\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu S, Gu L, Wu N, Song J, Yan J, Yang S, \u003cem\u003eet al.\u003c/em\u003e Overexpression of DTL enhances cell motility and promotes tumor metastasis in cervical adenocarcinoma by inducing RAC1-JNK-FOXO1 axis. Cell Death Dis 2021, 12(10): 929.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCockman ME, Masson N, Mole DR, Jaakkola P, Chang GW, Clifford SC, \u003cem\u003eet al.\u003c/em\u003e Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein. The Journal of biological chemistry 2000, 275(33): 25733\u0026ndash;25741.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen YC, Chen IS, Huang GJ, Kang CH, Wang KC, Tsao MJ, \u003cem\u003eet al.\u003c/em\u003e Targeting DTL induces cell cycle arrest and senescence and suppresses cell growth and colony formation through TPX2 inhibition in human hepatocellular carcinoma cells. Onco Targets Ther 2018, 11: 1601\u0026ndash;1616.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWicks EE, Semenza GL. Hypoxia-inducible factors: cancer progression and clinical translation. J Clin Invest 2022, 132(11).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLlovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, \u003cem\u003eet al.\u003c/em\u003e Sorafenib in advanced hepatocellular carcinoma. The New England journal of medicine 2008, 359(4): 378\u0026ndash;390.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang W, Chen Z, Zhang W, Cheng Y, Zhang B, Wu F, \u003cem\u003eet al.\u003c/em\u003e The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects. Signal Transduct Target Ther 2020, 5(1): 87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGwak GY, Yoon JH, Kim KM, Lee HS, Chung JW, Gores GJ. Hypoxia stimulates proliferation of human hepatoma cells through the induction of hexokinase II expression. J Hepatol 2005, 42(3): 358\u0026ndash;364.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng M, Wang Y, Bi L, Zhang P, Wang H, Zhao Z, \u003cem\u003eet al.\u003c/em\u003e CRL4A(DTL) degrades DNA-PKcs to modulate NHEJ repair and induce genomic instability and subsequent malignant transformation. Oncogene 2021, 40(11): 2096\u0026ndash;2111.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Wang R, Qiu C, Cao C, Zhang J, Ge J, \u003cem\u003eet al.\u003c/em\u003e Role of DTL in Hepatocellular Carcinoma and Its Impact on the Tumor Microenvironment. Front Immunol 2022, 13: 834606.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePan HW, Chou HY, Liu SH, Peng SY, Liu CL, Hsu HC. Role of L2DTL, cell cycle-regulated nuclear and centrosome protein, in aggressive hepatocellular carcinoma. Cell Cycle 2006, 5(22): 2676\u0026ndash;2687.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReig M, Forner A, Rimola J, Ferrer-Fabrega J, Burrel M, Garcia-Criado A, \u003cem\u003eet al.\u003c/em\u003e BCLC strategy for prognosis prediction and treatment recommendation: The 2022 update. J Hepatol 2022, 76(3): 681\u0026ndash;693.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLlovet JM, Zucman-Rossi J, Pikarsky E, Sangro B, Schwartz M, Sherman M, \u003cem\u003eet al.\u003c/em\u003e Hepatocellular carcinoma. Nature reviews Disease primers 2016, 2: 16018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuagliata L, Quintavalle C, Lanzafame M, Matter MS, Novello C, di Tommaso L, \u003cem\u003eet al.\u003c/em\u003e High expression of HOXA13 correlates with poorly differentiated hepatocellular carcinomas and modulates sorafenib response in in vitro models. Laboratory investigation; a journal of technical methods and pathology 2018, 98(1): 95\u0026ndash;105.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia P, Zhang H, Xu K, Jiang X, Gao M, Wang G, \u003cem\u003eet al.\u003c/em\u003e MYC-targeted WDR4 promotes proliferation, metastasis, and sorafenib resistance by inducing CCNB1 translation in hepatocellular carcinoma. Cell Death Dis 2021, 12(7): 691.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL.Harris A. HYPOXIA \u0026mdash; A KEY REGULATORY FACTOR IN TUMOUR GROWTH. Nat Rev Cancer 2002 Jan, 2(1):38\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa Z, Wang LZ, Cheng JT, Lam WST, Ma X, Xiang X, \u003cem\u003eet al.\u003c/em\u003e Targeting Hypoxia-Inducible Factor-1-Mediated Metastasis for Cancer Therapy. Antioxidants \u0026amp; redox signaling 2021, 34(18): 1484\u0026ndash;1497.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDou C, Zhou Z, Xu Q, Liu Z, Zeng Y, Wang Y, \u003cem\u003eet al.\u003c/em\u003e Hypoxia-induced TUFT1 promotes the growth and metastasis of hepatocellular carcinoma by activating the Ca(2+)/PI3K/AKT pathway. Oncogene 2019, 38(8): 1239\u0026ndash;1255.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong Z, Liu T, Chen J, Ge C, Zhao F, Zhu M, \u003cem\u003eet al.\u003c/em\u003e HIF-1alpha-induced RIT1 promotes liver cancer growth and metastasis and its deficiency increases sensitivity to sorafenib. Cancer Lett 2019, 460: 96\u0026ndash;107.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSemenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003, 3(10): 721\u0026ndash;732.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang MH, Mohan CD, Deivasigamani A, Chinnathambi A, Alharbi SA, Rangappa KS, \u003cem\u003eet al.\u003c/em\u003e Procaine Abrogates the Epithelial-Mesenchymal Transition Process through Modulating c-Met Phosphorylation in Hepatocellular Carcinoma. Cancers 2022, 14(20).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGurzu S, Kobori L, Fodor D, Jung I. Epithelial Mesenchymal and Endothelial Mesenchymal Transitions in Hepatocellular Carcinoma: A Review. \u003cem\u003eBioMed research international\u003c/em\u003e 2019, 2019: 2962580.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo M, Niu Y, Xie M, Liu X, Li X. Notch signaling, hypoxia, and cancer. Frontiers in Oncology 2023, 13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArtavanis-Tsakonas S, Muskavitch MA. Notch: the past, the present, and the future. Current topics in developmental biology 2010, 92: 1\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao L, Qi L, Zhang L, Song W, Yu Y, Xu C, \u003cem\u003eet al.\u003c/em\u003e Human nonsense-mediated RNA decay regulates EMT by targeting the TGF-\u0026szlig; signaling pathway in lung adenocarcinoma. Cancer Lett 2017, 403: 246\u0026ndash;259.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMackintosh C, Ord\u0026oacute;\u0026ntilde;ez JL, Garc\u0026iacute;a-Dom\u0026iacute;nguez DJ, Sevillano V, Llombart-Bosch A, Szuhai K, \u003cem\u003eet al.\u003c/em\u003e 1q gain and CDT2 overexpression underlie an aggressive and highly proliferative form of Ewing sarcoma. Oncogene 2012, 31(10): 1287\u0026ndash;1298.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3691309/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3691309/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDenticleless E3 ubiquitin protein ligase homolog (DTL), the substrate receptor of the CRL4A complex, plays a central role in genome stability. Even though the oncogenic function of DTL has been investigated in several cancers, its specific role in Hepatocellular Carcinoma (HCC) still needs further elucidation. Data from a clinical cohort (n\u0026thinsp;=\u0026thinsp;209), RNA-sequencing, and public database (TCGA and GEO) were analyzed, indicating that DTL is closely related to patient prognosis and could serve as a promising prognostic indicator in HCC. Functionally, DTL promoted the proliferation, metastasis, and sorafenib resistance of HCC in vitro. In the orthotopic tumor transplantation and tail vein injection model, DTL promoted the growth and metastasis of HCC in vivo. Mechanically, we revealed for the first time that DTL was transcriptionally activated by hypoxia-inducible factor 1α (HIF-1α) under hypoxia and functioned as a downstream effector molecule of HIF-1α. DTL facilitates HCC cell proliferation, metastasis, and epithelial-mesenchymal transition through the Notch pathway. These results suggested that DTL may be a potential biomarker and therapeutic target for HCC.\u003c/p\u003e","manuscriptTitle":"Hypoxia-induced DTL promotes the proliferation, metastasis, and sorafenib resistance of Hepatocellular Carcinoma through Notch Signaling Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 19:15:54","doi":"10.21203/rs.3.rs-3691309/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-04-15T13:32:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-28T19:07:24+00:00","index":4,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-11T02:08:51+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-03-09T10:20:41+00:00","index":4,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-02-19T15:33:17+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-01-12T09:15:31+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-01-04T10:03:39+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-01-04T09:38:44+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-01-04T09:29:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-12-01T11:24:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2023-12-01T08:30:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-01T08:30:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"17730492-e036-46b2-8c7c-566307f22408","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":27945624,"name":"Biological sciences/Cancer/Oncogenes"},{"id":27945625,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Liver cancer"},{"id":27945626,"name":"Biological sciences/Cancer/Metastasis"}],"tags":[],"updatedAt":"2024-10-10T07:06:17+00:00","versionOfRecord":{"articleIdentity":"rs-3691309","link":"https://doi.org/10.1038/s41419-024-07089-4","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2024-10-09 04:00:00","publishedOnDateReadable":"October 9th, 2024"},"versionCreatedAt":"2024-01-08 19:15:54","video":"","vorDoi":"10.1038/s41419-024-07089-4","vorDoiUrl":"https://doi.org/10.1038/s41419-024-07089-4","workflowStages":[]},"version":"v1","identity":"rs-3691309","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3691309","identity":"rs-3691309","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}