The DDX39B/FUT3/TGFβR-I Axis Promotes Tumor Metastasis and EMT in Colorectal Cancer

Abstract Background: DDX39B is a member of the DEAD box (DDX) RNA helicase family required for nearly all cellular RNA metabolism processes. The exact role and potential molecular mechanism of DDX39B in the progression of human colorectal cancer (CRC) remain to be investigated. Methods: Western blotting and quantitative real-time PCR (qRT-PCR) were conducted to detect the expression of DDX39B in CRC tissues and cell lines. Transwell and wound healing assays were conducted to assess the migration and invasion ability of CRC cells with DDX39B overexpressed or silencing. Orthotopic transplantation model of nude mice was performed to validate CRC metastasis in vivo. RNA sequencing (RNA-seq) and RNA binding protein immunoprecipitation (RIP) assay verified the direct regulation of DDX39B on the splicing and nuclear export of FUT3 mRNA, cytoplasmic and nuclear RNA isolation confirmed the nuclear export effect of DDX39B on FUT3. qRT-PCR was conducted to quantify FUT3 splicing variants. Lectin blotting was conducted to evaluate the fucosylation level of TGFβR-I.Results: In the present study, we demonstrate that DDX39B expression was higher in CRC tissues than in adjacent normal tissues. Gain- and loss- of- function assays revealed that DDX39B facilitated the metastasis of CRC in vivo and in vitro. Mechanistically, RNA-seq and RIP showed that DDX39B upregulated FUT3 expression by binding the first exon of FUT3 mRNA, which promote the mRNA splicing and export of FUT3. RNA-seq results and qRT-PCR showed that overexpression of DDX39B may favor the longer FUT3 mRNA products that contain the complete and longer exon 2, suggesting an alternative splicing of FUT3. Upregulation of FUT3 accelerated the fucosylation of TGFβR-I, thus activating the TGFβ/SMAD signaling pathway, eventually driving the epithelial-mesenchymal transition (EMT) program and contributing to CRC progression. Conclusions: Our finding demonstrated for the first time that the DDX39B/FUT3/TGFβR-I axis promotes the progression of CRC. These findings not only provide new insight into the role of DDX39B in mRNA splicing and export and tumorigenesis, but also shed light on the effect of aberrant fucosylation on CRC progression.

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RBM24 Mediates Lymph Node Metastasis and Epithelial-Mesenchymal-Transition in Human Hypopharyngeal Squamous Cell Carcinoma Via Regulating Twist1

10.21203/rs.3.rs-1167348/v1 ◽

2021 ◽

Author(s):

Yuhong Liu ◽

Tao Lu ◽

Min Pan ◽

Dan Yu ◽

Yanshi Li ◽

...

Keyword(s):

Lymph Node ◽

Rna Binding ◽

Epithelial Mesenchymal Transition ◽

Migration And Invasion ◽

Inhibitory Effects ◽

Mesenchymal Transition ◽

Qrt Pcr ◽

Node Metastasis ◽

Fadu Cells

Abstract Background: Hypopharyngeal squamous cell carcinoma (HSCC) has the worst prognosis among head and neck tumours, and Lymph node (LN) metastasis mainly accounts for the poor prognosis. RBM24 (RNA Binding Motif Protein 24) regulates target RNA as an RNA binding protein involved in several cancers. However, its role in HSCC remains completely unknown. Here we attempt to explore the effects of RBM24 on HSCC. Methods: RNA sequencing was conducted to find the differentially expressed genes in tumour tissues from HSCC patients with LN metastasis and without LN metastasis in our previous study. Expression of RBM24 in HSCC tissues was analyzed by qRT-PCR, western blot and immunohistochemistry. Cell proliferation was tested by CCK8 assay as well as Colony formation analysis. Cell migration and invasion capacity were estimated by transwell assay. The wound healing assay was also carried out to evaluate the motility of FaDu cells. QRT-PCR, western blot and immunofluorescence assays were conducted to detect the process of EMT. A popliteal lymph node metastasis model was constructed to explore the effect of RBM24 on HSCC in vivo.Results: RBM24 was remarkably down-regulated in HSCC patients with LN metastasis, and low expression of RBM24 was inextricably linked to the poor prognosis. Knockdown of RBM24 facilitated the proliferation, migration and invasion of RBM24, whereas overexpression of RBM24 showed the opposite effects and suppressed the epithelial-mesenchymal-transition (EMT) process. Overexpression of Twist1 could reverse the inhibitory effects of RBM24 on motility and invasion of FaDu cells. The inhibitory effects of RBM24 on tumour growth and lymphatic metastasis in HSCC were demonstrated by the in vivo experiment as well.Conclusions: These results indicated RBM24 was a suppressor gene and might inhibit EMT and LN metastasis in HSCC via regulating Twist1.

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The DDX39B/FUT3/TGFβR-I axis promotes tumor metastasis and EMT in colorectal cancer

Cell Death and Disease ◽

10.1038/s41419-020-03360-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chengcheng He ◽

Aimin Li ◽

Qiuhua Lai ◽

Jian Ding ◽

Qun Yan ◽

...

Keyword(s):

Colorectal Cancer ◽

Rna Binding ◽

Epithelial Mesenchymal Transition ◽

Mrna Splicing ◽

Loss Of Function ◽

Mesenchymal Transition ◽

Normal Tissues ◽

Cytoplasmic Rna

AbstractDDX39B is a member of the DEAD box (DDX) RNA helicase family required for nearly all cellular RNA metabolic processes. The exact role and potential molecular mechanism of DDX39B in the progression of human colorectal cancer (CRC) remain to be investigated. In the present study, we demonstrate that DDX39B expression is higher in CRC tissues than in adjacent normal tissues. Gain- and loss-of-function assays revealed that DDX39B facilitates CRC metastasis in vivo and in vitro. Mechanistically, RNA-sequencing (RNA-seq) and RNA-binding protein immunoprecipitation-sequencing (RIP-seq) showed that DDX39B binds directly to the FUT3 pre-mRNA and upregulates FUT3 expression. Splicing experiments in vitro using a Minigene assay confirmed that DDX39B promotes FUT3 pre-mRNA splicing. A nuclear and cytoplasmic RNA separation assay indicates that DDX39B enhances the mRNA export of FUT3. Upregulation of FUT3 accelerates the fucosylation of TGFβR-I, which activates the TGFβ signaling pathway and eventually drives the epithelial–mesenchymal transition (EMT) program and contributes to CRC progression. These findings not only provide new insight into the role of DDX39B in mRNA splicing and export as well as in tumorigenesis, but also shed light on the effects of aberrant fucosylation on CRC progression.

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Long non-coding RNA H19 promotes colorectal cancer metastasis via binding to hnRNPAA2B1

10.21203/rs.3.rs-19097/v1 ◽

2020 ◽

Author(s):

Yu-Hui Zhang ◽

Wei-Bin Huang ◽

Yu-Jie Yuan ◽

Jin Li ◽

Jing Wu ◽

...

Keyword(s):

Colorectal Cancer ◽

Cancer Metastasis ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Qrt Pcr ◽

Non Coding Rna ◽

Potential Biomarker ◽

Long Non Coding Rna

Abstract Background Long non-coding RNA H19 was demonstrated to be significantly correlated with tumor metastasis. However, the specific functions of H19 in colorectal cancer (CRC) metastasis and the underlying mechanism are still largely unclear. Methods Use public database to screen the potential lncRNA crucial for metastasis in colorectal cancer. The expression of H19 in clinical CRC specimens was detected by qRT-PCR. The effect of H19 on the metastasis of CRC cells was investigated by transwell, wound healing assays, CCK-8 assays and animal studies. The potential proteins binding to H19 was identified by LC-MS and verified by RNA immunoprecipitation (RIP). The expression of indicated RNA and proteins were measured by qRT-PCR or western blot. Results We found the expression of lncRNA H19 was significantly upregulated in primary tumor and metastatic tissues, correlated with poor prognosis in CRC. Ectopic H19 expression promoted the metastasis of colorectal cancer cells in vitro and in vivo , and induced epithelial-to-mesenchymal transition (EMT). Mechanistically, H19 directly bound to hnRNPA2B1. Knockdown of hnRNPA2B1 attenuated the H19-induce migration and invasion in CRC cells. Furthermore, H19 stabilized and upregulated the expression of Raf-1 by facilitated the interaction between hnRNPA2B1 and Raf-1 mRNA, resulting in activation of Raf-ERK signaling. Conclusions Our findings demonstrate the role of H19/hnRNPA2B1/EMT axis in regulation CRC metastasis, suggested H19 could be a potential biomarker to predict prognosis as well as a therapeutic strategy for CRC.

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CSN8 is a key regulator in hypoxia-induced epithelial–mesenchymal transition and dormancy of colorectal cancer cells

Molecular Cancer ◽

10.1186/s12943-020-01285-4 ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Songwen Ju ◽

Feng Wang ◽

Yirong Wang ◽

Songguang Ju

Keyword(s):

Colorectal Cancer ◽

Cancer Cells ◽

Cancer Progression ◽

Epithelial Mesenchymal Transition ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Treatment Conditions ◽

Colorectal Cancer Cells

AbstractHypoxic stress plays a pivotal role in cancer progression; however, how hypoxia drives tumors to become more aggressive or metastatic and adaptive to adverse environmental stress is still poorly understood. In this study, we revealed that CSN8 might be a key regulatory switch controlling hypoxia-induced malignant tumor progression. We demonstrated that the expression of CSN8 increased significantly in colorectal cancerous tissues, which was correlated with lymph node metastasis and predicted poor patient survival. CSN8 overexpression induces the epithelial-mesenchymal transition (EMT) process in colorectal cancer cells, increasing migration and invasion. CSN8 overexpression arrested cell proliferation, upregulated key dormancy marker (NR2F1, DEC2, p27) and hypoxia response genes (HIF-1α, GLUT1), and dramatically enhanced survival under hypoxia, serum deprivation, or chemo-drug 5-fluorouracil treatment conditions. In particular, silenced CSN8 blocks the EMT and dormancy processes induced by the hypoxia of 1% O2 in vitro and undermines the adaptive capacity of colorectal cancer cells in vivo. The further study showed that CSN8 regulated EMT and dormancy partly by activating the HIF-1α signaling pathway, which increased HIF-1α mRNA expression by activating NF-κB and stabilized the HIF-1α protein via HIF-1α de-ubiquitination. Taken together, CSN8 endows primary colorectal cancer cells with highly aggressive/metastatic and adaptive capacities through regulating both EMT and dormancy induced by hypoxia. CSN8 could serve as a novel prognostic biomarker for colorectal cancer and would be an ideal target of disseminated dormant cell elimination and tumor metastasis, recurrence, and chemoresistance prevention.

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KLK8 promotes the proliferation and metastasis of colorectal cancer via the activation of EMT associated with PAR1

Cell Death and Disease ◽

10.1038/s41419-021-04149-x ◽

2021 ◽

Vol 12 (10) ◽

Author(s):

Qing Hua ◽

Zhirong Sun ◽

Yi Liu ◽

Xuefang Shen ◽

Weiwei Zhao ◽

...

Keyword(s):

Colorectal Cancer ◽

Nude Mice ◽

Positive Impact ◽

Xenograft Model ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Metastasis Model ◽

Sw480 Cells

AbstractKallikrein-related peptidase 8 (KLK8) acts as an oncogene or anti-oncogene in various tumours, and the abnormal expression of KLK8 is involved in the carcinogenesis of several tumours. However, the role of KLK8 in colorectal cancer (CRC) and the underlying mechanism remain largely unclear. In this study, the carcinogenic effect of KLK8 was determined via CCK-8 and colony formation assays in vitro and a xenograft model in nude mice in vivo. The metastasis-promoting effect of KLK8 was investigated with transwell migration and invasion assays and wound-healing assay in vitro and a metastasis model in nude mice in vivo. Bioinformatics analyses and mechanistic experiments were conducted to elucidate the molecular mechanism. Herein, we reported that KLK8 had a promotive effect on the proliferation, migration and invasion of RKO and SW480 cells. Epithelial−mesenchymal transition (EMT) played an important role in the promotive effects of KLK8 on CRC. In addition, protease-activated receptor-1 (PAR-1) antagonist SCH79797 but not protease-activated receptor-2 (PAR-2) antagonist FSLLRY-NH2 attenuated the proliferation, migration and invasion of KLK8-upregulated RKO and SW480 cells. PAR-1 antagonist SCH79797 reduced the tumour volume of xenograft model and decreased the metastatic nodules in the livers of metastasis model. Furthermore, SCH79797 could reverse the positive impact of KLK8 on the EMT process in CRC both in vitro and in vivo. Taken together, these findings demonstrated for the first time that KLK8 promoted EMT and CRC progression, and this effect might be, at least partly mediated by PAR1-dependent pathway.

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LINC00460 / DHX9/IGF2BP2 complex promotes colorectal cancer proliferation and metastasis by mediating HMGA1 mRNA stability depending on m6A modification

10.21203/rs.3.rs-103647/v1 ◽

2020 ◽

Author(s):

Pingfu Hou ◽

Sen Meng ◽

Minle Li ◽

Tian Lin ◽

Sufang Chu ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Proliferation ◽

Molecular Mechanism ◽

Mrna Stability ◽

Functional Characterization ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Hmga1 Expression

Abstract Background: Increasing studies have shown that long noncoding RNAs (lncRNAs) are pivotal regulators participating in carcinogenic progression and tumor metastasis in colorectal cancer (CRC). Although lncRNA long intergenic noncoding RNA 460 (LINC00460) has been reported in CRC, the role and molecular mechanism of LINC00460 in CRC progression still requires exploration.Methods: The expression levels of LINC00460 were analyzed by using a tissue microarray containing 498 CRC tissues and their corresponding non-tumor adjacent tissues. The correlations between the LINC00460 expression level and clinicopathological features were evaluated. The functional characterization of the role and molecular mechanism of LINC00460 in CRC was investigated through a series of in vitro and in vivo experiments.Results: LINC00460 expression was increased in human CRC, and high LINC00460 expression was correlated with poor five-year overall survival and disease-free survival. LINC00460 overexpression sufficiently induced the epithelial–mesenchymal transition and promoted tumor cell proliferation, migration, and invasion in vitro and tumor growth and metastasis in vivo. In addition, LINC00460 enhanced the protein expression of high-mobility group AT-hook 1 (HMGA1) by directly interacting with IGF2BP2 and DHX9 to bind the 3′ untranslated region (UTR) of HMGA1 mRNA and increased the stability of HMGA1 mRNA. In addition, the N6-methyladenosine (m6A) modification of HMGA1 mRNA by METTL3 enhanced HMGA1 expression in CRC. Finally, it suggested that HMGA1 was essential for LINCC046-induced cell proliferation, migration, and invasion.Conclusions: LINC00460 may be a novel oncogene of CRC through interacting with IGF2BP2 and DHX9 and bind to the m6A modified HMGA1 mRNA to enhance the HMGA1 mRNA stability. LINC00460 can serve as a promising predictive biomarker for the diagnosis and prognosis among patients with CRC.

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RNA-binding protein CELF1 enhances cell migration, invasion, and chemoresistance by targeting ETS2 in colorectal cancer

Clinical Science ◽

10.1042/cs20191174 ◽

2020 ◽

Vol 134 (14) ◽

pp. 1973-1990

Author(s):

Huaiming Wang ◽

Rongkang Huang ◽

Wentai Guo ◽

Xiusen Qin ◽

Zifeng Yang ◽

...

Keyword(s):

Colorectal Cancer ◽

Rna Binding ◽

Malignant Tumors ◽

Binding Protein ◽

Rna Binding Protein ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Tumor Growth Model

Abstract Colorectal cancer (CRC) is often diagnosed at later stages after it has metastasized to other organs. The development of chemoresistance also contributes to a poor prognosis. Therefore, an increased understanding of the metastatic properties of CRC and chemoresistance could improve patient survival. CUGBP elav-like family member 1 (CELF1) is an RNA-binding protein, which is overexpressed in many human malignant tumors. However, the influence of CELF1 in CRC is unclear. V-ets erythroblastosis virus E26 oncogene homologue 2 (ETS2) is an evolutionarily conserved proto-oncogene known to be overexpressed in a variety of human cancers including CRC. In thespresent tudy, we investigated the association between CELF1 and ETS2 in CRC tumorigenesis and oxaliplatin (L-OHP) resistance. We found a positive correlation between the elevated expression of CELF1 and ETS2 in human CRC tissues. Overexpression of CELF1 increased CRC cell proliferation, migration, and invasion in vitro and in a xenograft tumor growth model in vivo, and induced resistance to L-OHP. In contrast, CELF1 knockdown improved the response of CRC cells to L-OHP. Overexpression of ETS2 increased the malignant behavior of CRC cells (growth, migration, and invasion) and L-OHP resistance in vitro. Moreover, L-OHP resistance induced by CELF1 overexpression was reversed by ETS2 knockdown. The results of luciferase reporter and ribonucleoprotein immunoprecipitation assays indicated that CELF1 up-regulates ETS2 by binding to its 3′-UTR. Taken together, our findings have identified that CELF1 regulates ETS2 in a mechanism that results in CRC tumorigenesis and L-OHP resistance, and CELF1 may be a promising target for overcoming chemoresistance in CRC.

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WNT4 secreted by tumor tissues promotes tumor progression in colorectal cancer by activation of the Wnt/β-catenin signalling pathway

10.21203/rs.3.rs-62264/v2 ◽

2020 ◽

Author(s):

Dongmei Yang ◽

Qing Li ◽

Renduo Shang ◽

Liwen Yao ◽

Lianlian Wu ◽

...

Keyword(s):

Colorectal Cancer ◽

Tumor Resection ◽

Signalling Pathway ◽

Tumor Xenograft ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Normal Tissues ◽

Potential Biomarker ◽

Wnt Ligands

Abstract Background: Wingless and Int-related protein (Wnt) ligands are aberrantly expressed in patients with colorectal cancer (CRC). However, the aberrant level of Wnt ligands in serum have not been explored. Here, we aimed to identify the levels of WNT4 in serum and explored its oncogenic role in CRC Methods: The Oncomine database was used to analyze the relationship between WNT4 and the prognosis of CRC. ELISA was performed to measure WNT4 levels in serum and conditioned medium from fresh CRC tissues and adjacent normal tissues. Western blot and immunohistochemistry were carried out to measure the expression of WNT4 in human CRC tissues and adjacent normal tissues. The migration and invasion of CRC cells were determined by trans-well assay, and the effects of WNT4 on CRC invasion and metastasis in vivo were verified by tumor xenograft in nude mice. Cancer-associated fibroblasts (CAFs) and angiogenesis in subcutaneous nodules were detected by immunofluorescence (IF). In addition, the suspended spheres formation and tube formation assay were performed to explore the effects of WNT4 on CAFs and angiogenesis respectively. Results: WNT4 was significantly upregulated in serum of CRC patients, and CRC tissues were identified as an important source of elevated WNT4 levels in CRC patients. Interestingly, elevated levels of WNT4 in serum were downregulated after tumor resection. Furthermore, we found that WNT4 contributed to epithelial-to-mesenchymal transition (EMT) and activated fibroblasts by activating the WNT4/β-catenin pathway in vitro and in vivo. Moreover, angiogenesis was induced via the WNT4/β-catenin/Ang2 pathway. Those effects could be reversed by ICG-001, a β-catenin/TCF inhibitor. Conclusion: Our findings indicated that serum levels of WNT4 may be a potential biomarker for CRC. WNT4 secreted by colorectal cancer tissues promote the progression of CRC by inducing EMT, activate fibroblasts and promote angiogenesis through the canonical Wnt/β-catenin signalling pathway.

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miR-125b Promotes Colorectal Cancer Migration and Invasion by Dual-Targeting CFTR and CGN

Cancers ◽

10.3390/cancers13225710 ◽

2021 ◽

Vol 13 (22) ◽

pp. 5710

Author(s):

Xiaohui Zhang ◽

Tingyu Li ◽

Ya-Nan Han ◽

Minghui Ge ◽

Pei Wang ◽

...

Keyword(s):

Colorectal Cancer ◽

Target Genes ◽

Epithelial Mesenchymal Transition ◽

Rho Kinase ◽

Causative Factor ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Mesenchymal Transition

Metastasis contributes to the poor prognosis of colorectal cancer, the causative factor of which is not fully understood. Previously, we found that miR-125b (Accession number: MIMAT0000423) contributed to cetuximab resistance in colorectal cancer (CRC). In this study, we identified a novel mechanism by which miR-125b enhances metastasis by targeting cystic fibrosis transmembrane conductance regulator (CFTR) and the tight junction-associated adaptor cingulin (CGN) in CRC. We found that miR-125b expression was upregulated in primary CRC tumors and metastatic sites compared with adjacent normal tissues. Overexpression of miR-125b in CRC cells enhanced migration capacity, while knockdown of miR-125b decreased migration and invasion. RNA-sequencing (RNA-seq) and dual-luciferase reporter assays identified CFTR and CGN as the target genes of miR-125b, and the inhibitory impact of CFTR and CGN on metastasis was further verified both in vitro and in vivo. Moreover, we found that miR-125b facilitated the epithelial-mesenchymal transition (EMT) process and the expression and secretion of urokinase plasminogen activator (uPA) by targeting CFTR and enhanced the Ras Homolog Family Member A (RhoA)/Rho Kinase (ROCK) pathway activity by targeting CGN. Together, these findings suggest miR-125b as a key functional molecule in CRC and a promising biomarker for the diagnosis and treatment of CRC.

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Mechanism and Molecular Network of RBM8A-Mediated Regulation of Oxaliplatin Resistance in Hepatocellular Carcinoma

Frontiers in Oncology ◽

10.3389/fonc.2020.585452 ◽

2021 ◽

Vol 10 ◽

Author(s):

Rong Liang ◽

Jinyan Zhang ◽

Zhihui Liu ◽

Ziyu Liu ◽

Qian Li ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Rna Binding ◽

Epithelial Mesenchymal Transition ◽

Migration And Invasion ◽

Binding Motif ◽

Mesenchymal Transition ◽

Regulatory Pathways ◽

Wide Range

RNA-binding motif protein 8A (RBM8A) is abnormally overexpressed in hepatocellular carcinoma (HCC) and involved in the epithelial-mesenchymal transition (EMT). The EMT plays an important role in the development of drug resistance, suggesting that RBM8A may be involved in the regulation of oxaliplatin (OXA) resistance in HCC. Here we examined the potential involvement of RBM8A and its downstream pathways in OXA resistance using in vitro and in vivo models. RBM8A overexpression induced the EMT in OXA-resistant HCC cells, altering cell proliferation, apoptosis, migration, and invasion. Moreover, whole-genome microarrays combined with bioinformatics analysis revealed that RBM8A has a wide range of transcriptional regulatory capabilities in OXA-resistant HCC, including the ability to regulate several important tumor-related signaling pathways. In particular, histone deacetylase 9 (HDAC9) emerged as an important mediator of RBM8A activity related to OXA resistance. These data suggest that RBM8A and its related regulatory pathways represent potential markers of OXA resistance and therapeutic targets in HCC.

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