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Zhao L, Zou X, Deng J, Sun B, Li Y, Zhao L, Zhao H, Zhang X, Yuan X, Zhao X, Zou F. hnRNPH1 maintains mitochondrial homeostasis by establishing NRF1/DRP1 retrograde signaling under mitochondrial stress. Cell Death Differ 2025; 32:118-133. [PMID: 38898233 PMCID: PMC11742414 DOI: 10.1038/s41418-024-01331-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2023] [Revised: 06/11/2024] [Accepted: 06/13/2024] [Indexed: 06/21/2024] Open
Abstract
Mitochondrial homeostasis is coordinated through communication between mitochondria and the nucleus. In response to stress, mitochondria generate retrograde signals to protect against their dysfunction by activating the expression of nuclear genes involved in metabolic reprogramming. However, the mediators associated with mitochondria-to-nucleus communication pathways remain to be clarified. Here, we identified that hnRNPH1 functions as a pivotal mediator of mitochondrial retrograde signaling to maintain mitochondrial homeostasis. hnRNPH1 accumulates in the nucleus following mitochondrial stress in a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent manner. Accordingly, hnRNPH1 interacts with the transcription factor NRF1 and binds to the DRP1 promoter, enhancing the transcription of DRP1. Furthermore, in the cytoplasm, hnRNPH1 directly interacts with DRP1 and enhances DRP1 Ser616 phosphorylation, thereby increasing DRP1 translocation to mitochondrial outer membranes and triggering mitochondrial fission. Collectively, our findings reveal a novel role for hnRNPH1 in the mitochondrial-nuclear communication pathway to maintain mitochondrial homeostasis under stress and suggest that it may be a potential target for mitochondrial dysfunction diseases.
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Affiliation(s)
- Lili Zhao
- Department of Targeting Therapy and Immunology and Laboratory of Animal Tumor Models, Cancer Center and State Key Laboratory of Biotherapy and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China
| | - Xiaotian Zou
- Department of Targeting Therapy and Immunology and Laboratory of Animal Tumor Models, Cancer Center and State Key Laboratory of Biotherapy and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China
| | - Jiaqiang Deng
- Department of Targeting Therapy and Immunology and Laboratory of Animal Tumor Models, Cancer Center and State Key Laboratory of Biotherapy and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China
| | - Bin Sun
- Department of Targeting Therapy and Immunology and Laboratory of Animal Tumor Models, Cancer Center and State Key Laboratory of Biotherapy and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China
| | - Yan Li
- Department of Targeting Therapy and Immunology and Laboratory of Animal Tumor Models, Cancer Center and State Key Laboratory of Biotherapy and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China
| | - Li Zhao
- Department of Targeting Therapy and Immunology and Laboratory of Animal Tumor Models, Cancer Center and State Key Laboratory of Biotherapy and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China
| | - Hong Zhao
- Department of Targeting Therapy and Immunology and Laboratory of Animal Tumor Models, Cancer Center and State Key Laboratory of Biotherapy and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China
| | - Xiao Zhang
- Department of Targeting Therapy and Immunology and Laboratory of Animal Tumor Models, Cancer Center and State Key Laboratory of Biotherapy and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China
| | - Xieyong Yuan
- Department of Targeting Therapy and Immunology and Laboratory of Animal Tumor Models, Cancer Center and State Key Laboratory of Biotherapy and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China
| | - Xudong Zhao
- Department of Targeting Therapy and Immunology and Laboratory of Animal Tumor Models, Cancer Center and State Key Laboratory of Biotherapy and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China.
| | - Fangdong Zou
- Department of Targeting Therapy and Immunology and Laboratory of Animal Tumor Models, Cancer Center and State Key Laboratory of Biotherapy and Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China.
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Hong L, Yang P, Zhang L, Liu X, Wei X, Xiao W, Yu Z, Zhang J, Peng Y, Wu X, Tang W, Zhi F, Li G, Li A, Lin J, Liu S, Zhang H, Xiang L, Wang J. The VAX2-LINC01189-hnRNPF signaling axis regulates cell invasion and migration in gastric cancer. Cell Death Discov 2023; 9:387. [PMID: 37865686 PMCID: PMC10590441 DOI: 10.1038/s41420-023-01688-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2023] [Revised: 09/28/2023] [Accepted: 10/13/2023] [Indexed: 10/23/2023] Open
Abstract
Transcription factors (TFs) and long noncoding RNAs (lncRNAs) contribute to gastric cancer (GC). However, the roles of TFs and lncRNAs in the invasion and metastasis of GC remain largely unknown. Here, we observed that the transcription factor VAX2 is significantly upregulated in GC cells and tissues and acts as an oncogene. Moreover, high VAX2 expression is associated with the advancement of tumors in GC. In terms of functionality, the enforced expression of VAX2 promotes the proliferation and metastasis of GC cells. Mechanistically, VAX2 specifically interacts with the LINC01189 promoter and represses LINC01189 transcription. Furthermore, LINC01189 exhibits significant downregulation in GC and functions as a suppressor gene. Functionally, it inhibits migratory and invasive abilities in GC cells. In the context of GC metastasis, VAX2 plays a role in modulating it by trans-repressing the expression of LINC01189. Additionally, LINC01189 binds to hnRNPF to enhance hnRNPF degradation through ubiquitination. The cooperation between LINC01189 and hnRNPF regulates GC cell invasion and migration. In addition, both VAX2 and hnRNPF are highly expressed, while LINC01189 is expressed in at low levels in GC tissues compared to normal gastric tissues. Our study suggests that VAX2 expression facilitates, while LINC01189 expression suppresses, metastasis and that the VAX2-LINC01189-hnRNPF axis plays a contributory role in GC development.
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Grants
- 81974448, 82073066, 82103152, 82103598, 82273354 National Natural Science Foundation of China (National Science Foundation of China)
- 81974448, 82073066, 82103152, 82103598, 82273354 National Natural Science Foundation of China (National Science Foundation of China)
- 81974448, 82073066, 82103152, 82103598, 82273354 National Natural Science Foundation of China (National Science Foundation of China)
- 81974448, 82073066, 82103152, 82103598, 82273354 National Natural Science Foundation of China (National Science Foundation of China)
- 81974448, 82073066, 82103152, 82103598, 82273354 National Natural Science Foundation of China (National Science Foundation of China)
- 81974448, 82073066, 82103152, 82103598, 82273354 National Natural Science Foundation of China (National Science Foundation of China)
- 81974448, 82073066, 82103152, 82103598, 82273354 National Natural Science Foundation of China (National Science Foundation of China)
- 81974448, 82073066, 82103152, 82103598, 82273354 National Natural Science Foundation of China (National Science Foundation of China)
- 81974448, 82073066, 82103152, 82103598, 82273354 National Natural Science Foundation of China (National Science Foundation of China)
- 81974448, 82073066, 82103152, 82103598, 82273354 National Natural Science Foundation of China (National Science Foundation of China)
- 81974448, 82073066, 82103152, 82103598, 82273354 National Natural Science Foundation of China (National Science Foundation of China)
- 2022A1515012464 Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)
- 2022A1515012464 Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)
- 2022A1515012464 Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)
- 2022A1515012464 Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)
- 2022A1515012464 Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)
- 2022A1515012464 Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)
- 2022A1515012464 Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)
- 2022A1515012464 Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)
- 2022A1515012464 Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)
- 2022A1515012464 Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)
- JCYJ20210324135005013 Shenzhen Science and Technology Innovation Commission
- JCYJ20210324135005013 Shenzhen Science and Technology Innovation Commission
- Science and Technology Project of Guangdong Province, 2017B20209003.
- Longgang District Science and Technology Innovation Bureau, LGKCYLWS2021000012, LGKCYLWS2022-005.
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Affiliation(s)
- Linjie Hong
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Ping Yang
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Luyu Zhang
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Xuehua Liu
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
- Department of Gastroenterology, Shunde Hospital, Southern Medical University, Foshan, 528300, China
| | - Xiangyang Wei
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Wushuang Xiao
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Zhen Yu
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Jieming Zhang
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Ying Peng
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Xiaosheng Wu
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Weimei Tang
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Fachao Zhi
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Guoxin Li
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Aimin Li
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Jianjiao Lin
- Department of Gastroenterology, The Second Affiliated Hospital, School of Medicine, The Chinese University of Hong Kong, Shenzhen & Longgang District People's Hospital of Shenzhen, Shenzhen, 518172, China
| | - Side Liu
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
- Department of Gastroenterology, The Second Affiliated Hospital, School of Medicine, The Chinese University of Hong Kong, Shenzhen & Longgang District People's Hospital of Shenzhen, Shenzhen, 518172, China
| | - Hui Zhang
- Department of Gastroenterology, The Affiliated Hexian Memorial Hospital of Southern Medical University, Guangzhou, 511400, China.
| | - Li Xiang
- Department of Gastroenterology, The Second Affiliated Hospital, School of Medicine, The Chinese University of Hong Kong, Shenzhen & Longgang District People's Hospital of Shenzhen, Shenzhen, 518172, China.
| | - Jide Wang
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
- Department of Gastroenterology, The Second Affiliated Hospital, School of Medicine, The Chinese University of Hong Kong, Shenzhen & Longgang District People's Hospital of Shenzhen, Shenzhen, 518172, China.
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Lu X, Li Y, Li Y, Zhang X, Shi J, Feng H, Gao Y, Yu Z. Advances of multi-omics applications in hepatic precancerous lesions and hepatocellular carcinoma: The role of extracellular vesicles. Front Mol Biosci 2023; 10:1114594. [PMID: 37006626 PMCID: PMC10060991 DOI: 10.3389/fmolb.2023.1114594] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2022] [Accepted: 03/06/2023] [Indexed: 03/18/2023] Open
Abstract
Due to the lack of distinct early symptoms and specific biomarkers, most patients with hepatocellular carcinoma (HCC) are usually diagnosed at advanced stages, rendering the treatment ineffective and useless. Therefore, recognition of the malady at precancerous lesions and early stages is particularly important for improving patient outcomes. The interest in extracellular vesicles (EVs) has been growing in recent years with the accumulating knowledge of their multiple cargoes and related multipotent roles in the modulation of immune response and tumor progression. By virtue of the rapid advancement of high-throughput techniques, multiple omics, including genomics/transcriptomics, proteomics, and metabolomics/lipidomics, have been widely integrated to analyze the role of EVs. Comprehensive analysis of multi-omics data will provide useful insights for discovery of new biomarkers and identification of therapeutic targets. Here, we review the attainment of multi-omics analysis to the finding of the potential role of EVs in early diagnosis and the immunotherapy in HCC.
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Affiliation(s)
- Xiaona Lu
- Department of Liver Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Yuyao Li
- Department of Liver Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Yue Li
- Department of Liver Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Xuemei Zhang
- Department of Liver Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Jia Shi
- Department of Liver Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Hai Feng
- Institute of Infectious Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
- *Correspondence: Hai Feng, ; Yueqiu Gao, ; Zhuo Yu,
| | - Yueqiu Gao
- Department of Liver Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
- Institute of Infectious Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
- *Correspondence: Hai Feng, ; Yueqiu Gao, ; Zhuo Yu,
| | - Zhuo Yu
- Department of Liver Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
- *Correspondence: Hai Feng, ; Yueqiu Gao, ; Zhuo Yu,
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Qureshi QUA, Audas TE, Morin RD, Coyle KM. Emerging roles for heterogeneous ribonuclear proteins in normal and malignant B cells. Biochem Cell Biol 2023; 101:160-171. [PMID: 36745874 DOI: 10.1139/bcb-2022-0332] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are among the most abundantly expressed RNA binding proteins in the cell and play major roles in all facets of RNA metabolism. hnRNPs are increasingly appreciated as essential for mammalian B cell development by regulating the carefully ordered expression of specific genes. Due to this tight regulation of the hnRNP-RNA network, it is no surprise that a growing number of genes encoding hnRNPs have been causally associated with the onset or progression of many cancers, including B cell neoplasms. Here we discuss our current understanding of hnRNP-driven regulation in normal, perturbed, and malignant B cells, and the most recent and emerging therapeutic innovations aimed at targeting the hnRNP-RNA network in lymphoma.
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Affiliation(s)
- Qurat Ul Ain Qureshi
- Department of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby, BC, Canada
| | - Timothy E Audas
- Department of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby, BC, Canada
| | - Ryan D Morin
- Department of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby, BC, Canada.,Canada's Michael Smith Genome Sciences Centre at BC Cancer, Vancouver, BC, Canada
| | - Krysta M Coyle
- Department of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby, BC, Canada
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Translational Regulation by hnRNP H/F Is Essential for the Proliferation and Survival of Glioblastoma. Cancers (Basel) 2022; 14:cancers14051283. [PMID: 35267591 PMCID: PMC8909726 DOI: 10.3390/cancers14051283] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Accepted: 02/21/2022] [Indexed: 02/05/2023] Open
Abstract
Deregulation of mRNA translation is a widespread characteristic of glioblastoma (GBM), aggressive malignant brain tumors that are resistant to conventional therapies. RNA-binding proteins (RBPs) play a critical role in translational regulation, yet the mechanisms and impact of these regulations on cancer development, progression and response to therapy remain to be fully understood. Here, we showed that hnRNP H/F RBPs are potent regulators of translation through several mechanisms that converge to modulate the expression and/or the activity of translation initiation factors. Among these, hnRNP H/F regulate the phosphorylation of eIF4E and its translational targets by controlling RNA splicing of the A-Raf kinase mRNA, which in turn modulates the MEK-ERK/MAPK signaling pathway. The underlying mechanism involves RNA G-quadruplex (RG4s), RNA structures whose modulation phenocopies hnRNP H/F translation regulation in GBM cells. Our results highlighted that hnRNP H/F are essential for key functional pathways regulating proliferation and survival of GBM, highlighting its targeting as a promising strategy for improving therapeutic outcomes.
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Chen X, Gong R, Wang J, Ma B, Lei K, Ren H, Wang J, Zhao C, Wang L, Yu Q. Identification of HnRNP Family as Prognostic Biomarkers in Five Major Types of Gastrointestinal Cancer. Curr Gene Ther 2022; 22:449-461. [PMID: 35794744 PMCID: PMC9906633 DOI: 10.2174/1566523222666220613113647] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Revised: 04/14/2022] [Accepted: 04/25/2022] [Indexed: 11/22/2022]
Abstract
BACKGROUND Heterogeneous nuclear ribonucleoproteins (hnRNPs), a large family of RNAbinding proteins, have been implicated in tumor progression in multiple cancer types. However, the expression pattern and prognostic value of hnRNPs in five gastrointestinal (GI) cancers, including gastric, colorectal, esophageal, liver, and pancreatic cancer, remain to be investigated. OBJECTIVE The current research aimed to identify prognostic biomarkers of the hnRNP family in five major types of gastrointestinal cancer. METHODS Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), and Kaplan-Meier Plotter were used to explore the hnRNPs expression levels concerning clinicopathological parameters and prognostic values. The protein level of hnRNPU was validated by immunohistochemistry (IHC) in human tissue specimens. Genetic alterations of hnRNPs were analyzed using cBioportal, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to illustrate the biological functions of co-expressed genes of hnRNPs. RESULTS The vast majority of hnRNPs were highly expressed in five types of GI cancer tissues compared to their adjacent normal tissues, and mRNA levels of hnRNPA2B1, D, Q, R, and U were significantly different in various GI cancer types at different stages. In addition, Kaplan-Meier analysis revealed that the increased hnRNPs expression levels were correlated with better prognosis in gastric and rectal cancer patients (log-rank p < 0.05). In contrast, patients with high levels of hnRNPs exhibited a worse prognosis in esophageal and liver cancer (log-rank p < 0.05). Using immunohistochemistry, we further confirmed that hnRNPU was overexpressed in gastric, rectal, and liver cancers. In addition, hnRNPs genes were altered in patients with GI cancers, and RNA-related processing was correlated with hnRNPs alterations. CONCLUSION We identified differentially expressed genes of hnRNPs in tumor tissues versus adjacent normal tissues, which might contribute to predicting tumor types, early diagnosis, and targeted therapies in five major types of GI cancer.
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Affiliation(s)
- Xianghan Chen
- Department of Pathology, School of Basic Medicine, Qingdao University, Qingdao 266071, China
- Center of Tumor Immunology and Cytotherapy, Medical Research Center of the Affiliated Hospital of Qingdao University, Qingdao 266000, China
| | - Ruining Gong
- Center of Tumor Immunology and Cytotherapy, Medical Research Center of the Affiliated Hospital of Qingdao University, Qingdao 266000, China
- Department of Gastroenterology, The Affiliated Hospital of Qingdao University, Qingdao 266000, China
| | - Jia Wang
- Center of Tumor Immunology and Cytotherapy, Medical Research Center of the Affiliated Hospital of Qingdao University, Qingdao 266000, China
| | - Boyi Ma
- Center of Tumor Immunology and Cytotherapy, Medical Research Center of the Affiliated Hospital of Qingdao University, Qingdao 266000, China
- Department of Gastroenterology, The Affiliated Hospital of Qingdao University, Qingdao 266000, China
| | - Ke Lei
- Center of Tumor Immunology and Cytotherapy, Medical Research Center of the Affiliated Hospital of Qingdao University, Qingdao 266000, China
| | - He Ren
- Center of Tumor Immunology and Cytotherapy, Medical Research Center of the Affiliated Hospital of Qingdao University, Qingdao 266000, China
- Department of Gastroenterology, The Affiliated Hospital of Qingdao University, Qingdao 266000, China
| | - Jigang Wang
- Department of Pathology, The Affiliated Hospital of Qingdao University, Qingdao 266000, China
| | - Chenyang Zhao
- Center of Tumor Immunology and Cytotherapy, Medical Research Center of the Affiliated Hospital of Qingdao University, Qingdao 266000, China
| | - Lili Wang
- Department of Pathology, The Affiliated Hospital of Qingdao University, Qingdao 266000, China
| | - Qian Yu
- Center of Tumor Immunology and Cytotherapy, Medical Research Center of the Affiliated Hospital of Qingdao University, Qingdao 266000, China
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Proteomic Characterization of Colorectal Cancer Tissue from Patients Identifies Novel Putative Protein Biomarkers. Curr Issues Mol Biol 2021; 43:1043-1056. [PMID: 34563043 PMCID: PMC8929084 DOI: 10.3390/cimb43020074] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2021] [Revised: 08/29/2021] [Accepted: 08/30/2021] [Indexed: 12/11/2022] Open
Abstract
Colorectal cancer (CRC) is one of the leading causes of cancer-related death over the world. There is a great need for biomarkers capable of early detection and as targets for treatment. Differential protein expression was investigated with two-dimensional gel electrophoresis (2D-PAGE) followed by identification with liquid chromatography–tandem mass spectrometry (LC-MS/MS) in CRC patient tissue from (i) the peripheral part of the tumor, (ii) the central part of the tumor as well as from (iii) a non-involved part of the colorectal tissue. The expression patterns of six identified proteins were further evaluated by one-dimensional Western blot (1D-WB) analysis of the CRC tissue. Proteins that were perturbed in expression level in the peripheral or in the central part of the tumor as compared with the non-involved part included S100A11, HNRNPF, HNRNPH1 or HNRNPH2, GSTP1, PKM and FABP1. These identified markers may have future diagnostic potential or may be novel treatment targets after further evaluation in larger patient cohorts.
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Abstract
Amyloid precursor protein (APP) is a transmembrane protein that plays a crucial role in the production of amyloid-β peptides. Any disruption in APP protein production, its mRNA decay rate or processing may result in abnormal production of amyloid-β peptides and subsequent development of protein aggregation diseases. Therefore, the equilibrium is crucial for neuronal function. An association study of heterogeneous nuclear ribonucleoprotein (hnRNP)-F and hnRNP H1 with APP was carried out in Neuro-2a (N2a) cells. In the present study, we found that hnRNP F and hnRNP H1 were significantly upregulated in the hippocampus of APP/PS1 mice. The changes in APP expression were positively associated with hnRNP F and hnRNP H1 when hnRNP F and hnRNP H1 were depleted or increased in N2a cells. Importantly, cross-linked RNA immunoprecipitation demonstrated binding affinities of hnRNP F and hnRNP H1 for App mRNA. Mechanistically, mRNA stability assay revealed that overexpression of hnRNP F or hnRNP H1 increases the APP level by stabilizing App mRNA half-life, implying that levels of hnRNP F and hnRNP H1 can change the production of APP. Further understanding of the regulatory mechanism of APP expression in association with hnRNP F and hnRNP H1 would provide insights into the mechanism underlying the maintenance of brain health and cognition. This study provides a theoretical basis for the development of hnRNP-stabilizing compounds to regulate APP.
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Affiliation(s)
- Muhammad I Khan
- Institute on Aging and Brain Disorders, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences
- Neurodegenerative Disease Research Center
- CAS Key Laboratory of Brain Function and Disease
| | - Juan Zhang
- Institute on Aging and Brain Disorders, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences
- Neurodegenerative Disease Research Center
- CAS Key Laboratory of Brain Function and Disease
| | - Qiang Liu
- Institute on Aging and Brain Disorders, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences
- Neurodegenerative Disease Research Center
- CAS Key Laboratory of Brain Function and Disease
- National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei
- CAS Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, China
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Nikolaou KC, Vatandaslar H, Meyer C, Schmid MW, Tuschl T, Stoffel M. The RNA-Binding Protein A1CF Regulates Hepatic Fructose and Glycerol Metabolism via Alternative RNA Splicing. Cell Rep 2020; 29:283-300.e8. [PMID: 31597092 DOI: 10.1016/j.celrep.2019.08.100] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2019] [Revised: 08/09/2019] [Accepted: 08/29/2019] [Indexed: 01/11/2023] Open
Abstract
The regulation of hepatic gene expression has been extensively studied at the transcriptional level; however, the control of metabolism through posttranscriptional gene regulation by RNA-binding proteins in physiological and disease states is less understood. Here, we report a major role for the hormone-sensitive RNA-binding protein (RBP) APOBEC1 complementation factor (A1CF) in the generation of hepatocyte-specific and alternatively spliced transcripts. Among these transcripts are isoforms for the dominant and high-affinity fructose-metabolizing ketohexokinase C and glycerol kinase, two key metabolic enzymes that are linked to hepatic gluconeogenesis and found to be markedly reduced upon hepatic ablation of A1cf. Consequently, mice lacking A1CF exhibit improved glucose tolerance and are protected from fructose-induced hyperglycemia, hepatic steatosis, and development of obesity. Our results identify a previously unreported function of A1CF as a regulator of alternative splicing of a subset of genes influencing hepatic glucose production through fructose and glycerol metabolism.
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Affiliation(s)
- Kostas C Nikolaou
- Institute of Molecular Health Sciences, ETH Zurich, Otto-Stern-Weg 7, 8093 Zürich, Switzerland
| | - Hasan Vatandaslar
- Institute of Molecular Health Sciences, ETH Zurich, Otto-Stern-Weg 7, 8093 Zürich, Switzerland
| | - Cindy Meyer
- Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
| | - Marc W Schmid
- MWSchmid GmbH, Möhrlistrasse 25, 8006 Zurich, Switzerland
| | - Thomas Tuschl
- Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
| | - Markus Stoffel
- Institute of Molecular Health Sciences, ETH Zurich, Otto-Stern-Weg 7, 8093 Zürich, Switzerland; Medical Faculty, University of Zurich, 8091 Zurich, Switzerland.
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10
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Pararajalingam P, Coyle KM, Arthur SE, Thomas N, Alcaide M, Meissner B, Boyle M, Qureshi Q, Grande BM, Rushton C, Slack GW, Mungall AJ, Tam CS, Agarwal R, Dawson SJ, Lenz G, Balasubramanian S, Gascoyne RD, Steidl C, Connors J, Villa D, Audas TE, Marra MA, Johnson NA, Scott DW, Morin RD. Coding and noncoding drivers of mantle cell lymphoma identified through exome and genome sequencing. Blood 2020; 136:572-584. [PMID: 32160292 PMCID: PMC7440974 DOI: 10.1182/blood.2019002385] [Citation(s) in RCA: 46] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2019] [Accepted: 02/20/2020] [Indexed: 12/11/2022] Open
Abstract
Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.
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Affiliation(s)
- Prasath Pararajalingam
- Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada
| | - Krysta M Coyle
- Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada
| | - Sarah E Arthur
- Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada
| | - Nicole Thomas
- Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada
| | - Miguel Alcaide
- Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada
| | - Barbara Meissner
- BC Cancer Centre for Lymphoid Cancer and
- BC Cancer Research Centre, Vancouver, BC, Canada
| | - Merrill Boyle
- BC Cancer Centre for Lymphoid Cancer and
- BC Cancer Research Centre, Vancouver, BC, Canada
| | - Quratulain Qureshi
- Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada
| | - Bruno M Grande
- Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada
| | - Christopher Rushton
- Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada
| | - Graham W Slack
- BC Cancer Centre for Lymphoid Cancer and
- BC Cancer Research Centre, Vancouver, BC, Canada
| | | | - Constantine S Tam
- Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
- University of Melbourne, Melbourne, VIC, Australia
| | - Rishu Agarwal
- Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
| | - Sarah-Jane Dawson
- Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
- University of Melbourne, Melbourne, VIC, Australia
| | - Georg Lenz
- Department of Medicine A, Hematology, Oncology, and Pneumology, University Hospital Münster, Münster, Germany
| | | | - Randy D Gascoyne
- BC Cancer Centre for Lymphoid Cancer and
- BC Cancer Research Centre, Vancouver, BC, Canada
| | - Christian Steidl
- BC Cancer Centre for Lymphoid Cancer and
- BC Cancer Research Centre, Vancouver, BC, Canada
| | - Joseph Connors
- BC Cancer Centre for Lymphoid Cancer and
- BC Cancer Research Centre, Vancouver, BC, Canada
| | - Diego Villa
- BC Cancer Centre for Lymphoid Cancer and
- BC Cancer Research Centre, Vancouver, BC, Canada
| | - Timothy E Audas
- Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada
| | - Marco A Marra
- BC Cancer Centre for Lymphoid Cancer and
- BC Cancer Research Centre, Vancouver, BC, Canada
| | | | - David W Scott
- BC Cancer Centre for Lymphoid Cancer and
- BC Cancer Research Centre, Vancouver, BC, Canada
| | - Ryan D Morin
- Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada
- Michael Smith Genome Sciences Centre, Vancouver, BC, Canada
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11
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Takahashi K, Fujiya M, Konishi H, Murakami Y, Iwama T, Sasaki T, Kunogi T, Sakatani A, Ando K, Ueno N, Kashima S, Moriichi K, Tanabe H, Okumura T. Heterogenous Nuclear Ribonucleoprotein H1 Promotes Colorectal Cancer Progression through the Stabilization of mRNA of Sphingosine-1-Phosphate Lyase 1. Int J Mol Sci 2020; 21:4514. [PMID: 32630435 PMCID: PMC7350029 DOI: 10.3390/ijms21124514] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2020] [Revised: 06/18/2020] [Accepted: 06/23/2020] [Indexed: 12/31/2022] Open
Abstract
The oncogenic properties of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) have been reported, although the tumor-promoting mechanism remains unclear. We herein report the mechanism underlying colorectal cancer cell progression mediated by hnRNP H1. The growth of colorectal cancer cells was suppressed by hnRNP H1 downregulation. A terminal deoxynucleotidyl transferase dUTP nick-end labeling assay revealed the anti-apoptotic effect of hnRNP H1 in colorectal cancer cells. An RNA immunoprecipitation assay revealed that hnRNP H1 bound to sphingosine-1-phosphate lyase 1 (SGPL1). Reverse transcription-polymerase chain reaction revealed the high expression of hnRNP H1 mRNA in colorectal cancer cells and Spearman's rank correlation coefficient showed a strong positive correlation between hnRNP H1 mRNA and SGPL1 mRNA. An siRNA of hnRNP H1 decreased SGPL1 mRNA expression in colorectal cancer cells, but not in non-tumorous cells. These findings suggested that hnRNP H1 increased SGPL1 mRNA expression specifically in cancer cells through direct binding. Targeted knockdown of hnRNP H1 or SGPL1 with siRNAs upregulated p53 phosphorylation and p53-associated molecules, resulting in cell growth inhibition, while hnRNP H1 upregulated the mRNA of SGPL1 and inhibited p53 activation, thereby promoting tumor cell growth. This is a novel mechanism underlying colorectal cancer cell progression mediated by hnRNP H1-SGPL1 mRNA stabilization.
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Affiliation(s)
- Keitaro Takahashi
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
| | - Mikihiro Fujiya
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
- Department of Gastroenterology and Advanced Medical Sciences, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan;
| | - Hiroaki Konishi
- Department of Gastroenterology and Advanced Medical Sciences, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan;
| | - Yuki Murakami
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
| | - Takuya Iwama
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
| | - Takahiro Sasaki
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
| | - Takehito Kunogi
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
| | - Aki Sakatani
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
- Department of Medicine, Knapp Center for Biomedical Discovery, The University of Chicago, 900 East 57th Street, 9th floor, Chicago, IL 60637, USA
| | - Katsuyoshi Ando
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
| | - Nobuhiro Ueno
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
| | - Shin Kashima
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
| | - Kentaro Moriichi
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
| | - Hiroki Tanabe
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
| | - Toshikatsu Okumura
- Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka-higashi, Asahikawa, Hokkaido 078-8510, Japan; (K.T.); (Y.M.); (T.I.); (T.S.); (T.K.); (A.S.); (K.A.); (N.U.); (S.K.); (K.M.); (H.T.); (T.O.)
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12
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Herviou P, Le Bras M, Dumas L, Hieblot C, Gilhodes J, Cioci G, Hugnot JP, Ameadan A, Guillonneau F, Dassi E, Cammas A, Millevoi S. hnRNP H/F drive RNA G-quadruplex-mediated translation linked to genomic instability and therapy resistance in glioblastoma. Nat Commun 2020; 11:2661. [PMID: 32461552 PMCID: PMC7253433 DOI: 10.1038/s41467-020-16168-x] [Citation(s) in RCA: 57] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2019] [Accepted: 04/14/2020] [Indexed: 01/01/2023] Open
Abstract
RNA G-quadruplexes (RG4s) are four-stranded structures known to control mRNA translation of cancer relevant genes. RG4 formation is pervasive in vitro but not in cellulo, indicating the existence of poorly characterized molecular machinery that remodels RG4s and maintains them unfolded. Here, we performed a quantitative proteomic screen to identify cytosolic proteins that interact with a canonical RG4 in its folded and unfolded conformation. Our results identified hnRNP H/F as important components of the cytoplasmic machinery modulating the structural integrity of RG4s, revealed their function in RG4-mediated translation and uncovered the underlying molecular mechanism impacting the cellular stress response linked to the outcome of glioblastoma. RNA G-quadruplexes (RG4s) have been functionally linked to cancer gene expression. Here, Herviou, Le Bras et al. have identified the protein machinery modulating RG4s and reveal the role and mechanism of hnRNP H/F and DHX36 in RG4-mediated translational regulation affecting cancer treatment in glioblastoma.
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Affiliation(s)
- Pauline Herviou
- Cancer Research Center of Toulouse (CRCT), INSERM UMR 1037, 31037, Toulouse, France.,Université Toulouse III Paul Sabatier, 31330, Toulouse, France.,Laboratoire d'Excellence "TOUCAN", Toulouse, France
| | - Morgane Le Bras
- Cancer Research Center of Toulouse (CRCT), INSERM UMR 1037, 31037, Toulouse, France.,Université Toulouse III Paul Sabatier, 31330, Toulouse, France.,Laboratoire d'Excellence "TOUCAN", Toulouse, France
| | - Leïla Dumas
- Cancer Research Center of Toulouse (CRCT), INSERM UMR 1037, 31037, Toulouse, France.,Université Toulouse III Paul Sabatier, 31330, Toulouse, France.,Laboratoire d'Excellence "TOUCAN", Toulouse, France
| | - Corinne Hieblot
- Cancer Research Center of Toulouse (CRCT), INSERM UMR 1037, 31037, Toulouse, France.,Université Toulouse III Paul Sabatier, 31330, Toulouse, France.,Laboratoire d'Excellence "TOUCAN", Toulouse, France
| | - Julia Gilhodes
- Institut Universitaire du Cancer de Toulouse-Oncopole, 31100, Toulouse, France
| | - Gianluca Cioci
- TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France
| | - Jean-Philippe Hugnot
- INSERM U1051, Institute for Neurosciences, Hôpital Saint Eloi, Université de Montpellier 2, 34090, Montpellier, France
| | - Alfred Ameadan
- Plateforme Protéomique 3P5, Université de Paris, Inserm U1016-institut Cochin, Labex GReX, 22 rue Méchain, 75014, Paris, France
| | - François Guillonneau
- Plateforme Protéomique 3P5, Université de Paris, Inserm U1016-institut Cochin, Labex GReX, 22 rue Méchain, 75014, Paris, France
| | - Erik Dassi
- Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento Via Sommarive 9, 38123, Trento, Italy.
| | - Anne Cammas
- Cancer Research Center of Toulouse (CRCT), INSERM UMR 1037, 31037, Toulouse, France. .,Université Toulouse III Paul Sabatier, 31330, Toulouse, France. .,Laboratoire d'Excellence "TOUCAN", Toulouse, France.
| | - Stefania Millevoi
- Cancer Research Center of Toulouse (CRCT), INSERM UMR 1037, 31037, Toulouse, France. .,Université Toulouse III Paul Sabatier, 31330, Toulouse, France. .,Laboratoire d'Excellence "TOUCAN", Toulouse, France.
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13
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Wall ML, Bera A, Wong FK, Lewis SM. Cellular stress orchestrates the localization of hnRNP H to stress granules. Exp Cell Res 2020; 394:112111. [PMID: 32473225 DOI: 10.1016/j.yexcr.2020.112111] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Revised: 05/19/2020] [Accepted: 05/21/2020] [Indexed: 12/01/2022]
Abstract
Heterogeneous nuclear ribonucleoprotein (hnRNP) H is a member of hnRNP H/F protein subfamily of hnRNPs that regulate the maturation and post-transcriptional processing of pre-mRNA. As a component of an mRNA export complex, hnRNP H shuttles mature mRNA from the nucleus to the cytoplasm. Although hnRNP H is primarily a nuclear protein, it can accumulate in the cytoplasm in certain tissues and cell types; however, the physiological relevance of hnRNP H cytoplasmic accumulation is unknown. Here we show that under cellular stress hnRNP H accumulates in the cytoplasm and is required for efficient recovery from cellular stress. Moreover, we find that cytoplasmic hnRNP H localizes to stress granules and that the RRM3 domain of hnRNP H is necessary for this localization. Together, our results demonstrate that hnRNP H accumulates in the cytoplasm under cellular stress and is recruited to stress granules.
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Affiliation(s)
- Michael L Wall
- Atlantic Cancer Research Institute, Moncton, New Brunswick, Canada
| | - Amit Bera
- Atlantic Cancer Research Institute, Moncton, New Brunswick, Canada
| | - Florence K Wong
- Atlantic Cancer Research Institute, Moncton, New Brunswick, Canada
| | - Stephen M Lewis
- Atlantic Cancer Research Institute, Moncton, New Brunswick, Canada; Department of Chemistry & Biochemistry, Université de Moncton, Moncton, New Brunswick, Canada; Senior Scientist, Beatrice Hunter Cancer Research Institute, Canada.
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14
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Xu C, Xie N, Su Y, Sun Z, Liang Y, Zhang N, Liu D, Jia S, Xing X, Han L, Li G, Tong T, Chen J. HnRNP F/H associate with hTERC and telomerase holoenzyme to modulate telomerase function and promote cell proliferation. Cell Death Differ 2019; 27:1998-2013. [PMID: 31863069 PMCID: PMC7244589 DOI: 10.1038/s41418-019-0483-6] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2019] [Revised: 12/09/2019] [Accepted: 12/10/2019] [Indexed: 12/16/2022] Open
Abstract
Human telomerase RNA component hTERC comprises multiple motifs that contribute to hTERC biogenesis, holoenzyme activity, and enzyme recruitment to telomeres. hTERC contains several guanine tracts (G-tracts) at its 5′-end, but its associated proteins and potential roles in telomerase function are still poorly understood. The heterogeneous nuclear ribonucleoproteins F, H1, and H2 (hnRNP F/H) are splicing factors that preferentially bind to poly(G)-rich sequences RNA. Here, we demonstrate that hnRNP F/H associate with both hTERC and telomerase holoenzyme to regulate telomerase activity. We reveal hnRNP F/H bind to the 5′-end region of hTERC in vitro and in vivo, and identify the first three G-tracts of hTERC and qRRM1 domain of hnRNP F/H are required for their interaction. Furthermore, hnRNP F/H also directly interact with telomerase holoenzyme. Functionally, we show that hnRNP F/H plays important roles in modulating telomerase activity and telomere length. Moreover, hnRNP F/H deletion greatly impair cancer and stem cell proliferation, and induce stem cell senescence, while hnRNP F/H overexpression delay stem cell senescence. Collectively, our findings unveil a novel role of hnRNP F/H as the binding partners of hTERC and telomerase holoenzyme to regulate telomerase function.
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Affiliation(s)
- Chenzhong Xu
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Nan Xie
- Department of Physiology and Pathophysiology, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Yuanyuan Su
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Zhaomeng Sun
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Yao Liang
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Na Zhang
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Doudou Liu
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Shuqin Jia
- Department of Molecular Diagnostics, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Beijing, 100142, China
| | - Xiaofang Xing
- Department of Molecular Diagnostics, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Beijing, 100142, China
| | - Limin Han
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Guodong Li
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Tanjun Tong
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Jun Chen
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China.
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15
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Yang T, An Z, Zhang C, Wang Z, Wang X, Liu Y, Du E, Liu R, Zhang Z, Xu Y. hnRNPM, a potential mediator of YY1 in promoting the epithelial-mesenchymal transition of prostate cancer cells. Prostate 2019; 79:1199-1210. [PMID: 31251827 DOI: 10.1002/pros.23790] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/15/2018] [Accepted: 02/22/2019] [Indexed: 12/11/2022]
Abstract
BACKGROUND With the popularity of serum prostate-specific antigen (PSA) screening, the number of newly diagnosed prostate cancer (PCa) patients is increasing. However, indolent or invasive PCa cannot be distinguished by PSA levels. Here, we mainly explored the role of heterogeneous nuclear ribonucleoprotein M (hnRNPM) in the invasiveness of PCa. METHODS Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis was used to detect the expressions of hnRNPM in PCa and benign prostate hyperplasia (BPH) tissues as well as in PCa cell lines. Immunohistochemistry was applied to detect the hnRNPM or Yin Yang 1 (YY1) expression in BPH, prostate adenocarcinoma (ADENO) and neuroendocrine prostate cancer (NEPC) tissues. After aberrant, the expression of hnRNPM in C4-2 and PC3 cells, the changes of cell migration and invasion were observed through wound-healing and transwell assays. We also predicted the transcription factor of hnRNPM through databases, then verified the association of hnRNPM and YY1 using chromatin immunoprecipitation (ChIP) and luciferase assays. RESULTS The expression level of hnRNPM is gradually reduced in BPH, ADENO, and NEPC tissues and it is less expressed in more aggressive PCa cell lines. Overexpression of hnRNPM can significantly reduce Twist1 expression, which inhibits the migration and invasion of PCa cells in vitro. In PCa cells, overexpression of YY1 can promote epithelial-mesenchymal transition by reducing hnRNPM expression. Furthermore, this effect caused by overexpression of YY1 can be partially attenuated by simultaneous overexpression of hnRNPM. CONCLUSIONS Our study demonstrates that hnRNPM negatively regulated PCa cell migration and invasion, and its expression can be transcriptionally inhibited by YY1. We speculated that hnRNPM may be a biomarker to assist in judging the aggressiveness of PCa.
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Affiliation(s)
- Tong Yang
- Department of Urology, Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, 23 Pingjiang Road, Hexi District, Tianjin, 300211, China
| | - Zesheng An
- Department of Urology, Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, 23 Pingjiang Road, Hexi District, Tianjin, 300211, China
| | - Changwen Zhang
- Department of Urology, Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, 23 Pingjiang Road, Hexi District, Tianjin, 300211, China
| | - Zhen Wang
- Department of Urology, Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, 23 Pingjiang Road, Hexi District, Tianjin, 300211, China
| | - Xiaoming Wang
- Department of Urology, Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, 23 Pingjiang Road, Hexi District, Tianjin, 300211, China
| | - Yan Liu
- Department of Urology, Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, 23 Pingjiang Road, Hexi District, Tianjin, 300211, China
| | - E Du
- Department of Urology, Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, 23 Pingjiang Road, Hexi District, Tianjin, 300211, China
| | - Ranlu Liu
- Department of Urology, Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, 23 Pingjiang Road, Hexi District, Tianjin, 300211, China
| | - Zhihong Zhang
- Department of Urology, Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, 23 Pingjiang Road, Hexi District, Tianjin, 300211, China
| | - Yong Xu
- Department of Urology, Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, 23 Pingjiang Road, Hexi District, Tianjin, 300211, China
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Tyson-Capper A, Gautrey H. Regulation of Mcl-1 alternative splicing by hnRNP F, H1 and K in breast cancer cells. RNA Biol 2018; 15:1448-1457. [PMID: 30468106 PMCID: PMC6333436 DOI: 10.1080/15476286.2018.1551692] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2018] [Revised: 10/10/2018] [Accepted: 11/02/2018] [Indexed: 01/27/2023] Open
Abstract
Myeloid cell leukemia-1 (Mcl -1) is one of the most frequently amplified genes in cancer, and its overexpression is associated with poor prognosis and drug resistance. As a member of the Bcl-2 family it is involved in the control of the mitochondrial (intrinsic) cell death pathway. Alternative splicing of the (Mcl-1) gene results in the expression of two functionally distinct proteins, the anti-apoptotic Mcl-1L (exon 2 included) and the pro-apoptotic Mcl-1S (exon 2 skipped). Our data shows that transfecting siRNAs that target hnRNP K and the hnRNP F/H family result in a switch in splicing towards the pro-apoptotic Mcl-1S. Specific binding sites for these and other Mcl-1 splicing factors were investigated and identified by RNA immunoprecipitation and through construction of a Mcl-1 minigene construct. Moreover, this study shows up to a 30 fold change in the levels of Mcl-1S can be achieved through double and triple knockdowns of the most significant RNA binding proteins involved in Mcl-1 splicing, as well as activation of the mitochondrial cell death pathway. Targeting the splicing process of Mcl-1 along with other apoptotic regulators provides an exciting new therapeutic target in cancer cells, and may provide a way to overcome therapy resistance.
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Affiliation(s)
- Alison Tyson-Capper
- Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
| | - Hannah Gautrey
- Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
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17
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Sofi S, Fitzgerald JC, Jähn D, Dumoulin B, Stehling S, Kuhn H, Ufer C. Functional characterization of naturally occurring genetic variations of the human guanine-rich RNA sequence binding factor 1 (GRSF1). Biochim Biophys Acta Gen Subj 2018; 1862:866-876. [DOI: 10.1016/j.bbagen.2017.12.008] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2017] [Revised: 12/12/2017] [Accepted: 12/22/2017] [Indexed: 10/18/2022]
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18
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Sun YL, Liu F, Liu F, Zhao XH. Protein and gene expression characteristics of heterogeneous nuclear ribonucleoprotein H1 in esophageal squamous cell carcinoma. World J Gastroenterol 2016; 22:7322-7331. [PMID: 27621578 PMCID: PMC4997634 DOI: 10.3748/wjg.v22.i32.7322] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2016] [Revised: 05/16/2016] [Accepted: 06/02/2016] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the expression characteristics of heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) mRNA and protein in cell lines and tissues of esophageal squamous cell carcinoma (ESCC).
METHODS Western blotting was used to assess the expression of HNRNPH1 protein in seven ESCC cell lines and 30 paired fresh tissue specimens. The subcellular localization of HNRNPH1 was determined by immunofluorescence in ESCC cells. The RNA sequencing data from 87 patients with ESCC were obtained from the cancer genome atlas (TCGA), and the expression and clinical characteristics analysis of different transcript variants of HNRNPH1 were evaluated in this dataset. In addition, immunohistochemistry was carried out to detect the expression of HNRNPH1 protein in 125 patients.
RESULTS The expression of HNRNPH1 protein varied across different ESCC cell lines. It was exclusively restricted to the nucleus of the ESCC cells. There are two transcript variants of the HNRNPH1 gene. Variant 1 was constitutively expressed, and its expression did not change during tumorigenesis. In contrast, levels of variant 2 were low in non-tumorous tissues and were dramatically increased in ESCC (P = 0.0026). The high levels of variant 2 were associated with poorer differentiated tumors (P = 0.0287). Furthermore, in paired fresh tissue specimens, HNRNPH1 protein was overexpressed in 73.3% (22/30) of neoplastic tissues. HNRNPH1 was significantly upregulated in ESCC, with strong staining in 43.2% (54/125) of tumor tissues and 22.4% (28/125) of matched non-cancerous tissues (P = 0.0005). Positive HNRNPH1 expression was significantly associated with poor tumor differentiation degree (P = 0.0337).
CONCLUSION The different alternative transcript variants of HNRNPH1 exhibited different expression changes during tumorigenesis. Its mRNA and protein were overexpressed in ESCC and associated with poorer differentiation of tumor cells. These findings highlight the potential of HNRNPH1 in the therapy and diagnosis of ESCC.
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MESH Headings
- Adult
- Aged
- Alternative Splicing
- Apoptosis
- Biomarkers, Tumor/genetics
- Biomarkers, Tumor/metabolism
- Carcinoma, Squamous Cell/genetics
- Carcinoma, Squamous Cell/metabolism
- Carcinoma, Squamous Cell/pathology
- Cell Line, Tumor
- Cell Proliferation
- Esophageal Neoplasms/genetics
- Esophageal Neoplasms/metabolism
- Esophageal Neoplasms/pathology
- Esophageal Squamous Cell Carcinoma
- Female
- Gene Expression Regulation, Neoplastic
- Heterogeneous-Nuclear Ribonucleoprotein Group F-H/genetics
- Heterogeneous-Nuclear Ribonucleoprotein Group F-H/metabolism
- Humans
- Male
- Middle Aged
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- RNA, Neoplasm/genetics
- RNA, Neoplasm/metabolism
- Up-Regulation
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19
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Yang Y, Jia D, Kim H, Abd Elmageed ZY, Datta A, Davis R, Srivastav S, Moroz K, Crawford BE, Moparty K, Thomas R, Hudson RS, Ambs S, Abdel-Mageed AB. Dysregulation of miR-212 Promotes Castration Resistance through hnRNPH1-Mediated Regulation of AR and AR-V7: Implications for Racial Disparity of Prostate Cancer. Clin Cancer Res 2015; 22:1744-56. [PMID: 26553749 DOI: 10.1158/1078-0432.ccr-15-1606] [Citation(s) in RCA: 71] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2015] [Accepted: 10/11/2015] [Indexed: 12/31/2022]
Abstract
PURPOSE The causes of disproportionate incidence and mortality of prostate cancer among African Americans (AA) remain elusive. The purpose of this study was to investigate the mechanistic role and assess clinical utility of the splicing factor heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) in prostate cancer progression among AA men. EXPERIMENTAL DESIGN We employed an unbiased functional genomics approach coupled with suppressive subtractive hybridization (SSH) and custom cDNA microarrays to identify differentially expressed genes in microdissected tumors procured from age- and tumor grade-matched AA and Caucasian American (CA) men. Validation analysis was performed in independent cohorts and tissue microarrays. The underlying mechanisms of hnRNPH1 regulation and its impact on androgen receptor (AR) expression and tumor progression were explored. RESULTS Aberrant coexpression of AR and hnRNPH1 and downregulation of miR-212 were detected in prostate tumors and correlate with disease progression in AA men compared with CA men. Ectopic expression of miR-212 mimics downregulated hnRNPH1 transcripts, which in turn reduced expression of AR and its splice variant AR-V7 (or AR3) in prostate cancer cells. hnRNPH1 physically interacts with AR and steroid receptor coactivator-3 (SRC-3) and primes activation of androgen-regulated genes in a ligand-dependent and independent manner. siRNA silencing of hnRNPH1 sensitized prostate cancer cells to bicalutamide and inhibited prostate tumorigenesis in vivo CONCLUSIONS Our findings define novel roles for hnRNPH1 as a putative oncogene, splicing factor, and an auxiliary AR coregulator. Targeted disruption of the hnRNPH1-AR axis may have therapeutic implications to improve clinical outcomes in patients with advanced prostate cancer, especially among AA men.
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Affiliation(s)
- Yijun Yang
- Department of Urology, Tulane University School of Medicine, New Orleans, Louisiana
| | - Dingwu Jia
- Department of Urology, Tulane University School of Medicine, New Orleans, Louisiana
| | - Hogyoung Kim
- Department of Urology, Tulane University School of Medicine, New Orleans, Louisiana
| | | | - Amrita Datta
- Department of Urology, Tulane University School of Medicine, New Orleans, Louisiana
| | - Rodney Davis
- Department of Urology, Tulane University School of Medicine, New Orleans, Louisiana
| | - Sudesh Srivastav
- Department of Biostatistics, Tulane University School of Public Health and Tropical Medicine, New Orleans, Louisiana
| | - Krzysztof Moroz
- Department of Pathology, Tulane University School of Medicine, New Orleans, Louisiana. Tulane Cancer Center, Tulane University School of Medicine, New Orleans, Louisiana
| | - Byron E Crawford
- Department of Pathology, Tulane University School of Medicine, New Orleans, Louisiana
| | - Krishnarao Moparty
- Department of Urology, Tulane University School of Medicine, New Orleans, Louisiana. Division of Urology, Southeast Louisiana Veterans Health Care System, New Orleans, Louisiana
| | - Raju Thomas
- Department of Urology, Tulane University School of Medicine, New Orleans, Louisiana. Tulane Cancer Center, Tulane University School of Medicine, New Orleans, Louisiana
| | - Robert S Hudson
- Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland
| | - Stefan Ambs
- Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland
| | - Asim B Abdel-Mageed
- Department of Urology, Tulane University School of Medicine, New Orleans, Louisiana. Tulane Cancer Center, Tulane University School of Medicine, New Orleans, Louisiana. Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana.
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20
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An S, Yang Y, Ward R, Liu Y, Guo XX, Xu TR. A-Raf: A new star of the family of raf kinases. Crit Rev Biochem Mol Biol 2015; 50:520-31. [PMID: 26508523 DOI: 10.3109/10409238.2015.1102858] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
The Ras-Raf-MEK-MAPK (mitogen-activated protein kinase)-signaling pathway plays a key role in the regulation of many cellular functions, including cell proliferation, differentiation and transformation, by transmitting signals from membrane receptors to various cytoplasmic and nuclear targets. One of the key components of this pathway is the serine/threonine protein kinase, Raf. The Raf family kinases (A-Raf, B-Raf and C-Raf) have been intensively studied since being identified in the early 1980s as retroviral oncogenes, especially with respect to the discovery of activating mutations of B-Raf in a large number of tumors which led to intensified efforts to develop drugs targeting Raf kinases. This also resulted in a rapid increase in our knowledge of the biological functions of the B-Raf and C-Raf isoforms, which may in turn be contrasted with the little that is known about A-Raf. The biological functions of A-Raf remain mysterious, although it appears to share some of the basic properties of the other two isoforms. Recently, emerging evidence has begun to reveal the functions of A-Raf, of which some are kinase-independent. These include the inhibition of apoptosis by binding to MST2, acting as safeguard against oncogenic transformation by suppressing extracellular signal-regulated kinases (ERK) activation and playing a role in resistance to Raf inhibitors. In this review, we discuss the regulation of A-Raf protein expression, and the roles of A-Raf in apoptosis and cancer, with a special focus on its role in resistance to Raf inhibitors. We also describe the scaffold functions of A-Raf and summarize the unexpected complexity of Raf signaling.
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Affiliation(s)
- Su An
- a Faculty of Life Science and Technology , Kunming University of Science and Technology , Kunming , Yunnan , China and
| | - Yang Yang
- a Faculty of Life Science and Technology , Kunming University of Science and Technology , Kunming , Yunnan , China and
| | - Richard Ward
- b Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow , Scotland , UK
| | - Ying Liu
- a Faculty of Life Science and Technology , Kunming University of Science and Technology , Kunming , Yunnan , China and
| | - Xiao-Xi Guo
- a Faculty of Life Science and Technology , Kunming University of Science and Technology , Kunming , Yunnan , China and
| | - Tian-Rui Xu
- a Faculty of Life Science and Technology , Kunming University of Science and Technology , Kunming , Yunnan , China and
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21
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Gautrey H, Jackson C, Dittrich AL, Browell D, Lennard T, Tyson-Capper A. SRSF3 and hnRNP H1 regulate a splicing hotspot of HER2 in breast cancer cells. RNA Biol 2015; 12:1139-51. [PMID: 26367347 DOI: 10.1080/15476286.2015.1076610] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Overexpression of the oncogene HER2 occurs in 20-30% of invasive breast cancer and is associated with poor prognosis. A number of different splice variants of HER2 have been identified which produce functionally different proteins. Previously these splice variants have been investigated separately, but in the present study we collectively look at the expression and regulation of a group of HER2 splice variants produced by a splicing hotspot. Initial investigation in a cohort of tumor samples showed large variations in HER2 variant expression between patient samples. RNA interference studies identified 2 splicing factors involved in the regulation of splicing within this region, hnRNP H1 and SRSF3. siRNA targeting hnRNP H1 increases levels of X5 and the oncogenic variant Δ16HER2. Furthermore RNA chromatography assays demonstrated binding of hnRNP H1 to RNA in this region. Additionally the proto-oncogene SRSF3 was also identified as an important regulator of splicing with SRSF3 knockdown resulting in changes in all the splice variants located at the hotspot. Most notably knockdown of SRSF3 resulted in a switch from the oncogenic Δ16HER2 to p100 which inhibits cell proliferation. Binding of SRSF3 to RNA within this region was also demonstrated by RNA chromatography and more specifically 2 SRSF3 binding sites were identified within exon 15. SRSF3 and hnRNP H1 are the first splicing factors identified which regulate the production of these functionally distinct HER2 splice variants and therefore maybe important for the regulation of HER2 signaling.
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Affiliation(s)
- Hannah Gautrey
- a Institute of Cellular Medicine; Faculty of Medical Sciences; Newcastle University ; Newcastle upon Tyne , UK
| | - Claire Jackson
- a Institute of Cellular Medicine; Faculty of Medical Sciences; Newcastle University ; Newcastle upon Tyne , UK
| | - Anna-Lena Dittrich
- a Institute of Cellular Medicine; Faculty of Medical Sciences; Newcastle University ; Newcastle upon Tyne , UK
| | - David Browell
- b Queen Elizabeth Hospital, Gateshead ; Tyne and Wear , UK
| | - Thomas Lennard
- c Northern Institute for Cancer Research; Faculty of Medical Sciences; Newcastle University ; Newcastle upon Tyne , UK
| | - Alison Tyson-Capper
- a Institute of Cellular Medicine; Faculty of Medical Sciences; Newcastle University ; Newcastle upon Tyne , UK
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22
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Yang B, Wang S, Zeng J, Zhang Y, Ruan X, Han W, Yin B, Yuan J, Qiang B, Ying W, Qian X, Peng X. Proteomic screening and identification of microRNA-128 targets in glioma cells. Proteomics 2015; 15:2602-17. [DOI: 10.1002/pmic.201400128] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2014] [Revised: 02/09/2015] [Accepted: 03/13/2015] [Indexed: 11/06/2022]
Affiliation(s)
- Bin Yang
- The State Key Laboratory of Medical Molecular Biology; Department of Molecular Biology and Biochemistry; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing P. R. China
| | - Shan Wang
- The State Key Laboratory of Medical Molecular Biology; Department of Molecular Biology and Biochemistry; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing P. R. China
| | - Jiawei Zeng
- The State Key Laboratory of Proteomics, Beijing Proteome Research Center; Beijing Institute of Radiation Medicine; Beijing P. R. China
| | - Yu Zhang
- The State Key Laboratory of Medical Molecular Biology; Department of Molecular Biology and Biochemistry; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing P. R. China
| | - Xiangbin Ruan
- The State Key Laboratory of Medical Molecular Biology; Department of Molecular Biology and Biochemistry; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing P. R. China
| | - Wei Han
- The State Key Laboratory of Medical Molecular Biology; Department of Molecular Biology and Biochemistry; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing P. R. China
| | - Bin Yin
- The State Key Laboratory of Medical Molecular Biology; Department of Molecular Biology and Biochemistry; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing P. R. China
| | - Jiangang Yuan
- The State Key Laboratory of Medical Molecular Biology; Department of Molecular Biology and Biochemistry; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing P. R. China
| | - Boqin Qiang
- The State Key Laboratory of Medical Molecular Biology; Department of Molecular Biology and Biochemistry; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing P. R. China
| | - Wantao Ying
- The State Key Laboratory of Proteomics, Beijing Proteome Research Center; Beijing Institute of Radiation Medicine; Beijing P. R. China
| | - Xiaohong Qian
- The State Key Laboratory of Proteomics, Beijing Proteome Research Center; Beijing Institute of Radiation Medicine; Beijing P. R. China
| | - Xiaozhong Peng
- The State Key Laboratory of Medical Molecular Biology; Department of Molecular Biology and Biochemistry; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College; Beijing P. R. China
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23
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Gupta SK, Kosti I, Plaut G, Pivko A, Tkacz ID, Cohen-Chalamish S, Biswas DK, Wachtel C, Waldman Ben-Asher H, Carmi S, Glaser F, Mandel-Gutfreund Y, Michaeli S. The hnRNP F/H homologue of Trypanosoma brucei is differentially expressed in the two life cycle stages of the parasite and regulates splicing and mRNA stability. Nucleic Acids Res 2013; 41:6577-94. [PMID: 23666624 PMCID: PMC3711420 DOI: 10.1093/nar/gkt369] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Trypanosomes are protozoan parasites that cycle between a mammalian host (bloodstream form) and an insect host, the Tsetse fly (procyclic stage). In trypanosomes, all mRNAs are trans-spliced as part of their maturation. Genome-wide analysis of trans-splicing indicates the existence of alternative trans-splicing, but little is known regarding RNA-binding proteins that participate in such regulation. In this study, we performed functional analysis of the Trypanosoma brucei heterogeneous nuclear ribonucleoproteins (hnRNP) F/H homologue, a protein known to regulate alternative splicing in metazoa. The hnRNP F/H is highly expressed in the bloodstream form of the parasite, but is also functional in the procyclic form. Transcriptome analyses of RNAi-silenced cells were used to deduce the RNA motif recognized by this protein. A purine rich motif, AAGAA, was enriched in both the regulatory regions flanking the 3′ splice site and poly (A) sites of the regulated genes. The motif was further validated using mini-genes carrying wild-type and mutated sequences in the 3′ and 5′ UTRs, demonstrating the role of hnRNP F/H in mRNA stability and splicing. Biochemical studies confirmed the binding of the protein to this proposed site. The differential expression of the protein and its inverse effects on mRNA level in the two lifecycle stages demonstrate the role of hnRNP F/H in developmental regulation.
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Affiliation(s)
- Sachin Kumar Gupta
- Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
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24
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Chettouh H, Fartoux L, Aoudjehane L, Wendum D, Clapéron A, Chrétien Y, Rey C, Scatton O, Soubrane O, Conti F, Praz F, Housset C, Rosmorduc O, Desbois-Mouthon C. Mitogenic insulin receptor-A is overexpressed in human hepatocellular carcinoma due to EGFR-mediated dysregulation of RNA splicing factors. Cancer Res 2013; 73:3974-86. [PMID: 23633480 DOI: 10.1158/0008-5472.can-12-3824] [Citation(s) in RCA: 92] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Insulin receptor (IR) exists as two isoforms resulting from the alternative splicing of IR pre-mRNA. IR-B promotes the metabolic effects of insulin, whereas IR-A rather signals proliferative effects. IR-B is predominantly expressed in the adult liver. Here, we show that the alternative splicing of IR pre-mRNA is dysregulated in a panel of 85 human hepatocellular carcinoma (HCC) while being normal in adjacent nontumor liver tissue. An IR-B to IR-A switch is frequently observed in HCC tumors regardless of tumor etiology. Using pharmacologic and siRNA approaches, we show that the autocrine or paracrine activation of the EGF receptor (EGFR)/mitogen-activated protein/extracellular signal-regulated kinase pathway increases the IR-A:IR-B ratio in HCC cell lines, but not in normal hepatocytes, by upregulating the expression of the splicing factors CUGBP1, hnRNPH, hnRNPA1, hnRNPA2B1, and SF2/ASF. In HCC tumors, there is a significant correlation between the expression of IR-A and that of splicing factors. Dysregulation of IR pre-mRNA splicing was confirmed in a chemically induced model of HCC in rat but not in regenerating livers after partial hepatectomy. This study identifies a mechanism responsible for the generation of mitogenic IR-A and provides a novel interplay between IR and EGFR pathways in HCC. Increased expression of IR-A during neoplastic transformation of hepatocytes could mediate some of the adverse effects of hyperinsulinemia on HCC.
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Affiliation(s)
- Hamza Chettouh
- INSERM UMR_S 938, Centre de Recherche Saint-Antoine; UPMC Univ Paris 06, UMR_S 938, Paris, France
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25
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Dong X, Yang M, Sun H, Lü J, Zheng Z, Li Z, Zhong L. Combined measurement of CA 15-3 with novel autoantibodies improves diagnostic accuracy for breast cancer. Onco Targets Ther 2013; 6:273-9. [PMID: 23569391 PMCID: PMC3615893 DOI: 10.2147/ott.s43122] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Objective CA 15-3 is a traditional biomarker for advanced breast cancer with limited sensitivity for early stage patients. In order to increase the sensitivity for early detection, in this study, we introduced novel tumor-associated autoantibodies that were measured concurrently with serum CA 15-3 to evaluate their diagnostic advantage in breast cancer. Methods We investigated a T7 breast cancer complementary deoxyribonucleic acid (cDNA) phage library for tumor-associated antigens using sera from normal and breast cancer patients. Identified novel tumor-associated antigens phage proteins were then used to develop enzyme-linked immunosorbent assays to measure corresponding autoantibodies in 150 breast cancer, 150 normal, and 40 other cancer (non breast) patient serum samples. Meanwhile, the same samples were measured for CA 15-3 concentrations. Receiver operating characteristic curve analysis was used to evaluate the predictive accuracies of single markers as well as combined markers. Results Sequencing analysis revealed that two phage-expressed proteins were within the open reading frame and had significant homology to proteins heterogeneous nuclear ribonucleoproteins F (hnRNPF) and ferritin heavy chain (FTH1). Autoantibodies against hnRNPF and FTH1 alone were significantly higher in patients than in control serum samples (P < 0.01), and the area under the curve for hnRNPF and FTH1 alone was 0.73 and 0.69, respectively. However, when the two autoantibody biomarkers were analyzed in combination with serum CA 15-3 values, the area under the curve increased to 0.93, and the optimal sensitivity and specificity became 89.3% and 93.8%, respectively. Further messenger ribonucleic acid (mRNA) analysis showed that hnRNPF and FTH1 were significantly upregulated in tumor tissues. Conclusion Our results indicated that combined serologic biomarkers of tumor-associated antigens with autoantibodies may improve the diagnostic accuracy of breast cancer.
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Affiliation(s)
- Xuejun Dong
- Molecular Medicine Center of Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University, Shaoxing, People's Republic of China
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26
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Global effects of subchronic treatment of microcystin-LR on rat splenetic protein levels. J Proteomics 2012; 77:383-93. [DOI: 10.1016/j.jprot.2012.09.012] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2012] [Revised: 08/28/2012] [Accepted: 09/12/2012] [Indexed: 11/17/2022]
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Zhang D, Tai LK, Wong LL, Putti TC, Sethi SK, Teh M, Koay ESC. Proteomic characterization of differentially expressed proteins in breast cancer: Expression of hnRNP H1, RKIP and GRP78 is strongly associated with HER-2/neu status. Proteomics Clin Appl 2012; 2:99-107. [PMID: 21136783 DOI: 10.1002/prca.200780099] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The human epidermal growth factor receptor type 2 (HER-2/neu) oncoprotein is overexpressed in about 30% of breast cancers and associates with metastatic phenotypes of breast tumours. Dissecting the HER-2/neu-modulated molecules in cancer will be helpful in elucidating the underlying molecular mechanisms of HER-2/neu-driven tumourigenesis. We investigated the differential proteome profiles between microdissected HER-2/neu-positive and -negative tumours and unambiguously identified 21 proteins with diverse biological functions by peptide sequencing and NCBInr database interrogation. Six proteins were up-regulated whereas 15 were down-regulated in the HER-2/neu-positive tumours. Differential expressions of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), 78 kDa glucose-regulated protein (GRP78/Bip) and Raf-1 kinase inhibitor protein (RKIP), which have not been previously reported as being linked to HER-2/neu signalling, were further verified. Immunohistochemical staining on tissue microarray sections demonstrated a positive correlation of hnRNP H1 (p = 0.008) and negative correlations of GRP78 and RKIP (p = 0.018 and 0.013, respectively) with HER-2/neu. Heregulin α1 enhanced hnRNP H1, but reduced GRP78 and RKIP expression in BT474 cells in a dose-dependent manner, providing evidence of crosstalk between HER-2/neu signalling and these modulators. Our studies have identified novel modulators that are likely to be intricately involved in HER-2/neu-driven tumour proliferation, invasion and metastasis.
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Affiliation(s)
- Daohai Zhang
- Department of Laboratory Medicine, National University Hospital, Singapore; Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
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28
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Sun Z, Biela LM, Hamilton KL, Reardon KF. Concentration-dependent effects of the soy phytoestrogen genistein on the proteome of cultured cardiomyocytes. J Proteomics 2012; 75:3592-604. [PMID: 22521270 DOI: 10.1016/j.jprot.2012.04.001] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2011] [Revised: 03/29/2012] [Accepted: 04/02/2012] [Indexed: 12/23/2022]
Abstract
The soy-derived phytoestrogen genistein (GEN) has received attention for its potential benefits on the cardiovascular system by providing direct protection to cardiomyocytes against pathophysiological stresses. Here, we employed a proteomic approach to study the concentration-dependent effects of GEN treatments on cardiomyocytes. Cultured HL-1 cardiomyocytes were treated with low (1μM) and high (50μM) concentrations of GEN. Proteins were pre-fractionated by sequential hydrophilic/hydrophobic extraction and both protein fractions from each treatment group were separated by 2D gel electrophoresis (2DE). Overall, approximately 2,700 spots were visualized on the 2D gels. Thirty-nine and 99 spots changed in volume relative to controls (p<0.05) following the low- and high-concentration GEN treatments, respectively. From these spots, 25 and 62 protein species were identified by ESI-MS/MS and Mascot database searching, respectively. Identified proteins were further categorized according to their functions and possible links to cardioprotection were discussed. MetaCore gene ontology analysis suggested that 1μM GEN significantly impacted the anti-apoptosis process, and that both the low and high concentrations of GEN influenced the glucose catabolic process and regulation of ATPase activity. This proteomics study provides the first global insight into the molecular events triggered by GEN treatment in cardiomyocytes.
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Affiliation(s)
- Zeyu Sun
- Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO 80523-1370, USA
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Su CC, Su JH, Lin JJ, Chen CC, Hwang WI, Huang HH, Wu YJ. An investigation into the cytotoxic effects of 13-acetoxysarcocrassolide from the soft coral Sarcophyton crassocaule on bladder cancer cells. Mar Drugs 2011; 9:2622-2642. [PMID: 22363243 PMCID: PMC3280580 DOI: 10.3390/md9122622] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2011] [Revised: 11/18/2011] [Accepted: 11/30/2011] [Indexed: 01/01/2023] Open
Abstract
Active compounds from natural products have been widely studied. The anti-tumor effects of 13-acetoxysarcocrassolide isolated from Formosan soft coral Sarcophyton crassocaule on bladder cancer cells were examined in this study. An MTT assay showed that 13-acetoxysarcocrassolide was cytotoxic to bladder female transitional cancer (BFTC) cells. We determined that the BFTC cells underwent cell death through apoptosis by flow cytometry. Due to the highly-migratory nature of the BFTC cells, the ability of 13-acetoxysarcocrassolide to stop their migration was assessed by a wound healing assay. To determine which proteins were affected in the BFTC cells upon treatment, a comparative proteomic analysis was performed. By LC-MS/MS analysis, we identified that 19 proteins were up-regulated and eight were down-regulated. Seven of the proteins were confirmed by western blotting analysis. This study reveals clues to the potential mechanism of the cytotoxic effects of 13-acetoxysarcocrassolide on BFTC cells. Moreover, it suggests that PPT1 and hnRNP F could be new biomarkers for bladder cancer. The results of this study are also helpful for the diagnosis, progression monitoring and therapeutic strategies of transitional cell tumors.
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Affiliation(s)
- Ching-Chyuan Su
- Antai Medical Care Cooperation Antai Tian-Sheng Memorial Hospital, Pingtung 92842, Taiwan;
| | - Jui-Hsin Su
- National Museum of Marine Biology and Aquarium, Pingtung 94446, Taiwan;
| | - Jen-Jie Lin
- Department of Beauty Science, Meiho University, Pingtung 91202, Taiwan; (J.-J.L.); (C.-C.C.)
| | - Cheng-Chi Chen
- Department of Beauty Science, Meiho University, Pingtung 91202, Taiwan; (J.-J.L.); (C.-C.C.)
| | - Wen-Ing Hwang
- Department of Food Science and Nutrition, Meiho University, Pingtung 91202, Taiwan;
| | - Han Hsiang Huang
- Department of Beauty Science, Meiho University, Pingtung 91202, Taiwan; (J.-J.L.); (C.-C.C.)
- Authors to whom correspondence should be addressed; or (Y.-J.W.); (H.H.H.); Tel.: +886-8-7799821 (ext. 8600) (Y.-J.W.); +886-8-7799821 (ext. 8647) (H.H.H.); Fax: +886-8-7793281 (H.H.H.)
| | - Yu-Jen Wu
- Department of Beauty Science, Meiho University, Pingtung 91202, Taiwan; (J.-J.L.); (C.-C.C.)
- Authors to whom correspondence should be addressed; or (Y.-J.W.); (H.H.H.); Tel.: +886-8-7799821 (ext. 8600) (Y.-J.W.); +886-8-7799821 (ext. 8647) (H.H.H.); Fax: +886-8-7793281 (H.H.H.)
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White R, Gonsior C, Bauer NM, Krämer-Albers EM, Luhmann HJ, Trotter J. Heterogeneous nuclear ribonucleoprotein (hnRNP) F is a novel component of oligodendroglial RNA transport granules contributing to regulation of myelin basic protein (MBP) synthesis. J Biol Chem 2011; 287:1742-54. [PMID: 22128153 DOI: 10.1074/jbc.m111.235010] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Myelin basic protein (MBP) is a major component of central nervous system (CNS) myelin. The absence of MBP results in the loss of almost all compact myelin in the CNS. MBP mRNA is sorted into RNA granules that are transported to the periphery of oligodendrocytes in a translationally inactive state. A central mediator of this transport process is the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 that binds to the cis-acting A2-response element in the 3'UTR of MBP mRNA. Recently, we found that activation of the Src family nonreceptor tyrosine kinase Fyn in oligodendrocytes leads to phosphorylation of hnRNP A2 and to increased translation of MBP mRNA. Here, we identify the RNA-binding protein hnRNP F as a novel component of MBP mRNA transport granules. It is associated with hnRNP A2 and MBP mRNA in cytoplasmic granular structures and is involved in post-transcriptional regulation of MBP expression. Fyn kinase activity results in phosphorylation of hnRNP F in the cytoplasm and its release from MBP mRNA and RNA granules. Our results define hnRNP F as a regulatory element of MBP expression in oligodendrocytes and imply an important function of hnRNP F in the control of myelin synthesis.
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Affiliation(s)
- Robin White
- Department of Biology, Molecular Cell Biology, Johannes Gutenberg University of Mainz, Bentzelweg 3, 55128 Mainz, Germany.
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31
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Di Giammartino DC, Nishida K, Manley JL. Mechanisms and consequences of alternative polyadenylation. Mol Cell 2011; 43:853-66. [PMID: 21925375 DOI: 10.1016/j.molcel.2011.08.017] [Citation(s) in RCA: 567] [Impact Index Per Article: 40.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2011] [Revised: 08/02/2011] [Accepted: 08/09/2011] [Indexed: 12/11/2022]
Abstract
Alternative polyadenylation (APA) is emerging as a widespread mechanism used to control gene expression. Like alternative splicing, usage of alternative poly(A) sites allows a single gene to encode multiple mRNA transcripts. In some cases, this changes the mRNA coding potential; in other cases, the code remains unchanged but the 3' UTR length is altered, influencing the fate of mRNAs in several ways, for example, by altering the availability of RNA binding protein sites and microRNA binding sites. The mechanisms governing both global and gene-specific APA are only starting to be deciphered. Here we review what is known about these mechanisms and the functional consequences of alternative polyadenylation.
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Proteomic analysis of anti-tumor effects of 11-dehydrosinulariolide on CAL-27 cells. Mar Drugs 2011; 9:1254-1272. [PMID: 21822415 PMCID: PMC3148502 DOI: 10.3390/md9071254] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2011] [Revised: 06/27/2011] [Accepted: 07/07/2011] [Indexed: 01/11/2023] Open
Abstract
The anti-tumor effects of 11-dehydrosinulariolide, an active ingredient isolated from soft coral Sinularia leptoclados, on CAL-27 cells were investigated in this study. In the MTT assay for cell proliferation, increasing concentrations of 11-dehydrosinulariolide decreased CAL-27 cell viability. When a concentration of 1.5 μg/mL of 11-dehydrosinulariolide was applied, the CAL-27 cells viability was reduced to a level of 70% of the control sample. The wound healing function decreased as the concentration of 11-dehydrosinulariolide increased. The results in this study indicated that treatment with 11-dehydrosinulariolide for 6 h significantly induced both early and late apoptosis of CAL-27 cells, observed by flow cytometric measurement and microscopic fluorescent observation. A comparative proteomic analysis was conducted to investigate the effects of 11-dehydrosinulariolide on CAL-27 cells at the molecular level by comparison between the protein profiling (revealed on a 2-DE map) of CAL-27 cells treated with 11-dehydrosinulariolide and that of CAL-27 cells without the treatment. A total of 28 differential proteins (12 up-regulated and 16 down-regulated) in CAL-27 cells treated with 11-dehydrosinulariolide have been identified by LC-MS/MS analysis. Some of the differential proteins are associated with cell proliferation, apoptosis, protein synthesis, protein folding, and energy metabolism. The results of this study provided clues for the investigation of biochemical mechanisms of the anti-tumor effects of 11-dehydrosinulariolide on CAL-27 cells and could be valuable information for drug development and progression monitoring of oral squamous cell carcinoma (OSCC).
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hnRNP F directs formation of an exon 4 minus variant of tumor-associated NADH oxidase (ENOX2). Mol Cell Biochem 2011; 357:55-63. [PMID: 21625959 DOI: 10.1007/s11010-011-0875-5] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2011] [Accepted: 05/06/2011] [Indexed: 12/27/2022]
Abstract
HUVEC or mouse 3T3 cells infected with SV-40 generate within 3 to 5 days post-infection an ENOX2 species corresponding to the exon-4 minus splice variant of a tumor-associated NADH oxidase (ENOX2 or tNOX) expressed at the cancer cell surface. This study was to seek evidence for splicing factors that might direct formation of the exon 4 minus ENOX2 splice variant. To determine if silencing of ENOX2 exon 4 occurs because of motifs located in exon 4, transfections were performed on MCF-10A (mammary non-cancer), BT-20 (mammary cancer), and HeLa (cervical cancer) cells using a GFP minigene construct containing either a constitutively spliced exon (albumin exon 2) or the alternatively spliced ENOX2 exon 4 between the two GFP halves. Removal of exon 4 from the processed RNA of the GFP minigene construct occurred with HeLa and to a lesser extent with BT-20 but not in non-cancer MCF-10A cells. The Splicing Rainbow Program was used to identify all of the possible hnRNPs binding sites of exon 4 of ENOX2. There are 8 Exonic Splicing Silencers (ESSs) for hnRNP binding in the exon 4 sequences. Each of these sites were mutated by site-directed mutagenesis to test if any were responsible for the splicing skip. Results showed MutG75 ESS mutation changed the GFP expression which is a sign of splicing silence, while other mutations did not. As MutG75 changed the ESS binding site for hnRNP F, this result suggests that hnRNP F directs formation of the exon 4 minus variant of ENOX2.
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34
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Katz Y, Wang ET, Airoldi EM, Burge CB. Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat Methods 2010; 7:1009-15. [PMID: 21057496 PMCID: PMC3037023 DOI: 10.1038/nmeth.1528] [Citation(s) in RCA: 999] [Impact Index Per Article: 66.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2010] [Accepted: 10/08/2010] [Indexed: 12/31/2022]
Abstract
Through alternative splicing, most human genes express multiple isoforms that often differ in function. To infer isoform regulation from high-throughput sequencing of cDNA fragments (RNA-seq), we developed the mixture-of-isoforms (MISO) model, a statistical model that estimates expression of alternatively spliced exons and isoforms and assesses confidence in these estimates. Incorporation of mRNA fragment length distribution in paired-end RNA-seq greatly improved estimation of alternative-splicing levels. MISO also detects differentially regulated exons or isoforms. Application of MISO implicated the RNA splicing factor hnRNP H1 in the regulation of alternative cleavage and polyadenylation, a role that was supported by UV cross-linking-immunoprecipitation sequencing (CLIP-seq) analysis in human cells. Our results provide a probabilistic framework for RNA-seq analysis, give functional insights into pre-mRNA processing and yield guidelines for the optimal design of RNA-seq experiments for studies of gene and isoform expression.
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Affiliation(s)
- Yarden Katz
- Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA,Department of Biology, MIT, Cambridge, Massachusetts, USA
| | - Eric T Wang
- Department of Biology, MIT, Cambridge, Massachusetts, USA,Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts, USA
| | - Edoardo M Airoldi
- Department of Statistics and FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA
| | - Christopher B Burge
- Department of Biology, MIT, Cambridge, Massachusetts, USA,Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA
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35
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Stark M, Bram EE, Akerman M, Mandel-Gutfreund Y, Assaraf YG. Heterogeneous nuclear ribonucleoprotein H1/H2-dependent unsplicing of thymidine phosphorylase results in anticancer drug resistance. J Biol Chem 2010; 286:3741-54. [PMID: 21068389 DOI: 10.1074/jbc.m110.163444] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Thymidine phosphorylase (TP) catalyzes the conversion of thymidine to thymine and 2-deoxyribose-1-phosphate. The latter plays an important role in induction of angiogenesis. As such, many human malignancies exhibit TP overexpression that correlates with increased microvessel density, formation of aggressive tumors, and dismal prognosis. Because TP is frequently overexpressed in cancer, pro-drugs were developed that utilize TP activity for their bioactivation to cytotoxic drugs. In this respect, TP is indispensable for the pharmacologic activity of the chemotherapeutic drug capecitabine, as it converts its intermediary metabolite 5'-deoxyfluorouridine to 5-fluorouracil. Thus, loss of TP function confers resistance to the prodrug capecitabine, currently used for the treatment of metastatic colorectal cancer and breast cancer. However, drug resistance phenomena may frequently emerge that compromise the pharmacologic activity of capecitabine. Deciphering the molecular mechanisms underlying resistance to TP-activated prodrugs is an important goal toward the overcoming of such drug resistance phenomena. Here, we discovered that lack of TP protein in drug-resistant tumor cells is due to unsplicing of its pre-mRNA. Advanced bioinformatics identified the family of heterogeneous nuclear ribonucleoproteins (hnRNP) H/F as candidate splicing factors potentially responsible for impaired TP splicing. Indeed, whereas parental cells lacked nuclear localization of hnRNPs H1/H2 and F, drug-resistant cells harbored marked levels of these splicing factors. Nuclear RNA immunoprecipitation experiments established a strong binding of hnRNP H1/H2 to TP pre-mRNA, hence implicating them in TP splicing. Moreover, introduction of hnRNP H2 into drug-sensitive parental cells recapitulated aberrant TP splicing and 5'-deoxyfluorouridine resistance. Thus, this is the first study identifying altered function of hnRNP H1/H2 in tumor cells as a novel determinant of aberrant TP splicing thereby resulting in acquired chemoresistance to TP-activated fluoropyrimidine anticancer drugs.
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Affiliation(s)
- Michal Stark
- The Fred Wyszkowski Cancer Research Laboratory, Technion-Israel Institute of Technology, Haifa 32000, Israel
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Abstract
The hnRNPs (heterogeneous nuclear ribonucleoproteins) are RNA-binding proteins with important roles in multiple aspects of nucleic acid metabolism, including the packaging of nascent transcripts, alternative splicing and translational regulation. Although they share some general characteristics, they vary greatly in terms of their domain composition and functional properties. Although the traditional grouping of the hnRNPs as a collection of proteins provided a practical framework, which has guided much of the research on them, this approach is becoming increasingly incompatible with current knowledge about their structural and functional divergence. Hence, we review the current literature to examine hnRNP diversity, and discuss how this impacts upon approaches to the classification of RNA-binding proteins in general.
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37
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Structural basis of G-tract recognition and encaging by hnRNP F quasi-RRMs. Nat Struct Mol Biol 2010; 17:853-61. [PMID: 20526337 DOI: 10.1038/nsmb.1814] [Citation(s) in RCA: 122] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2010] [Accepted: 03/22/2010] [Indexed: 01/19/2023]
Abstract
The heterogeneous nuclear ribonucleoprotein (hnRNP) F is involved in the regulation of mRNA metabolism by specifically recognizing G-tract RNA sequences. We have determined the solution structures of the three quasi-RNA-recognition motifs (qRRMs) of hnRNP F in complex with G-tract RNA. These structures show that qRRMs bind RNA in a very unusual manner, with the G-tract 'encaged', making the qRRM a novel RNA binding domain. We defined a consensus signature sequence for qRRMs and identified other human qRRM-containing proteins that also specifically recognize G-tract RNAs. Our structures explain how qRRMs can sequester G-tracts, maintaining them in a single-stranded conformation. We also show that isolated qRRMs of hnRNP F are sufficient to regulate the alternative splicing of the Bcl-x pre-mRNA, suggesting that hnRNP F would act by remodeling RNA secondary and tertiary structures.
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38
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Zhang T, Huang XH, Dong L, Hu D, Ge C, Zhan YQ, Xu WX, Yu M, Li W, Wang X, Tang L, Li CY, Yang XM. PCBP-1 regulates alternative splicing of the CD44 gene and inhibits invasion in human hepatoma cell line HepG2 cells. Mol Cancer 2010; 9:72. [PMID: 20361869 PMCID: PMC2864215 DOI: 10.1186/1476-4598-9-72] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2009] [Accepted: 04/02/2010] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND PCBP1 (or alpha CP1 or hnRNP E1), a member of the PCBP family, is widely expressed in many human tissues and involved in regulation of transcription, transportation process, and function of RNA molecules. However, the role of PCBP1 in CD44 variants splicing still remains elusive. RESULTS We found that enforced PCBP1 expression inhibited CD44 variants expression including v3, v5, v6, v8, and v10 in HepG2 cells, and knockdown of endogenous PCBP1 induced these variants splicing. Invasion assay suggested that PCBP1 played a negative role in tumor invasion and re-expression of v6 partly reversed the inhibition effect by PCBP1. A correlation of PCBP1 down-regulation and v6 up-regulation was detected in primary HCC tissues. CONCLUSIONS We first characterized PCBP1 as a negative regulator of CD44 variants splicing in HepG2 cells, and loss of PCBP1 in human hepatic tumor contributes to the formation of a metastatic phenotype.
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Affiliation(s)
- Tong Zhang
- Beijing Institute of Radiation Medicine, Beijing, 100850, China
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A glycine-rich domain of hnRNP H/F promotes nucleocytoplasmic shuttling and nuclear import through an interaction with transportin 1. Mol Cell Biol 2010; 30:2552-62. [PMID: 20308327 DOI: 10.1128/mcb.00230-09] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Heterogeneous nuclear ribonucleoprotein (hnRNP) H and F are members of a closely related subfamily of hnRNP proteins that are implicated in many aspects of RNA processing. hnRNP H and F are alternative splicing factors for numerous U2- and U12-dependent introns. The proteins have three RNA binding domains and two glycine-rich domains and localize to both the nucleus and cytoplasm, but little is known about which domains govern subcellular localization or splicing activity. We show here that the central glycine-tyrosine-arginine-rich (GYR) domain is responsible for nuclear localization, and a nonclassical nuclear localization signal (NLS) was mapped to a short, highly conserved sequence whose activity was compromised by point mutations. Glutathione S-transferase (GST) pulldown assays demonstrated that the hnRNP H NLS interacts with the import receptor transportin 1. Finally, we show that hnRNP H/F are transcription-dependent shuttling proteins. Collectively, the results suggest that hnRNP H and F are GYR domain-dependent shuttling proteins whose posttranslational modifications may alter nuclear localization and hence function.
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40
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Rauch J, O'Neill E, Mack B, Matthias C, Munz M, Kolch W, Gires O. Heterogeneous nuclear ribonucleoprotein H blocks MST2-mediated apoptosis in cancer cells by regulating A-Raf transcription. Cancer Res 2010; 70:1679-88. [PMID: 20145135 DOI: 10.1158/0008-5472.can-09-2740] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signaling by activating the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway. Low kinase activity of A-Raf toward MEK suggested that A-Raf might have alternative functions. Here, we show that A-Raf prevents cancer cell apoptosis contingent on the expression of the heterogeneous nuclear ribonucleoprotein H (hnRNP H) splice factor, which is required for the correct transcription and expression of a-raf. Apoptosis was prevented by A-Raf through sequestration and inactivation of the proapoptotic MST2 kinase. Small interfering RNA-mediated knockdown of hnRNP H or A-Raf resulted in MST2-dependent apoptosis. In contrast, enforced expression of either hnRNP H or A-Raf partially counteracted apoptosis induced by etoposide. In vivo expression studies of colon specimens corroborated the overexpression of hnRNP H in malignant tissues and its correlation with A-Raf levels. Our findings define a novel mechanism that is usurped in tumor cells to escape naturally imposed apoptotic signals.
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Affiliation(s)
- Jens Rauch
- Department of Head and Neck Research, Ludwig-Maximilians-Universität München, D-81377 Munich, Germany
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41
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Exploring the biochemical mechanisms of cytotoxic gold compounds: a proteomic study. J Biol Inorg Chem 2010; 15:573-82. [DOI: 10.1007/s00775-010-0624-3] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2009] [Accepted: 12/28/2009] [Indexed: 10/19/2022]
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42
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Lee JH, Kim SH, Pascua PNQ, Song MS, Baek YH, Jin X, Choi JK, Kim CJ, Kim H, Choi YK. Direct interaction of cellular hnRNP-F and NS1 of influenza A virus accelerates viral replication by modulation of viral transcriptional activity and host gene expression. Virology 2009; 397:89-99. [PMID: 19926108 DOI: 10.1016/j.virol.2009.10.041] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2009] [Revised: 09/18/2009] [Accepted: 10/27/2009] [Indexed: 01/01/2023]
Abstract
To investigate novel NS1-interacting proteins, we conducted a yeast two-hybrid analysis, followed by co-immunoprecipitation assays. We identified heterogeneous nuclear ribonucleoprotein F (hnRNP-F) as a cellular protein interacting with NS1 during influenza A virus infection. Co-precipitation assays suggest that interaction between hnRNP-F and NS1 is a common and direct event among human or avian influenza viruses. NS1 and hnRNP-F co-localize in the nucleus of host cells, and the RNA-binding domain of NS1 directly interacts with the GY-rich region of hnRNP-F determined by GST pull-down assays with truncated proteins. Importantly, hnRNP-F expression levels in host cells indicate regulatory role on virus replication. hnRNP-F depletion by small interfering RNA (siRNA) shows 10- to 100-fold increases in virus titers corresponding to enhanced viral RNA polymerase activity. Our results delineate novel mechanism of action by which NS1 accelerates influenza virus replication by modulating normal cellular mRNA processes through direct interaction with cellular hnRNP-F protein.
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Affiliation(s)
- Jun Han Lee
- College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong Heungduk-Ku, Cheongju 361-763, Republic of Korea
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Xiao X, Wang Z, Jang M, Nutiu R, Wang ET, Burge CB. Splice site strength-dependent activity and genetic buffering by poly-G runs. Nat Struct Mol Biol 2009; 16:1094-100. [PMID: 19749754 PMCID: PMC2766517 DOI: 10.1038/nsmb.1661] [Citation(s) in RCA: 92] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2009] [Accepted: 07/21/2009] [Indexed: 12/29/2022]
Abstract
Pre-mRNA splicing is regulated through combinatorial activity of RNA motifs including splice sites and splicing regulatory elements (SREs). Here, we show that the activity of the G-run class of SREs is ∼4-fold higher when adjacent to intermediate strength 5'ss relative to weak 5'ss, and ∼1.3-fold higher relative to strong 5'ss. This dependence on 5'ss strength was observed in splicing reporters and in global microarray and mRNA-Seq analyses of splicing changes following RNAi against heterogeneous nuclear ribonucleoprotein (hnRNP) H, which crosslinked to G-runs adjacent to many regulated exons. An exon’s responsiveness to changes in hnRNP H levels therefore depends in a complex way on G-run abundance and 5'ss strength, and other splicing factors may function similarly. This pattern of activity enables G-runs and hnRNP H to buffer the effects of 5'ss mutations, augmenting the frequency of 5'ss polymorphism and the evolution of new splicing patterns.
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Affiliation(s)
- Xinshu Xiao
- Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
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44
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Fox JT, Stover PJ. Mechanism of the internal ribosome entry site-mediated translation of serine hydroxymethyltransferase 1. J Biol Chem 2009; 284:31085-96. [PMID: 19734143 DOI: 10.1074/jbc.m109.035576] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
The 5'-untranslated region (UTR) of serine hydroxymethyltransferase 1 (SHMT1) contains an internal ribosome entry site (IRES) that regulates SHMT1 expression, a rate-limiting enzyme in de novo thymidylate biosynthesis. In this study, we show that the SHMT1 IRES is the first example of a cellular IRES that is poly(A) tail-independent. Interactions between the 5'-UTR and 3'-UTR functionally replaced interactions between the poly(A) tail and the poly(A)-binding protein (PABP) to achieve maximal IRES-mediated translational efficiency. Depletion of the SHMT1 IRES-specific trans-acting factor (ITAF) CUG-binding protein 1 (CUGBP1) from in vitro translation extracts or deletion of the CUGBP1 binding site on the 3'-UTR of the SHMT1 transcript decreased the IRES activity of non-polyadenylylated biscistronic mRNAs relative to polyadenylylated biscistronic mRNAs and resulted in a requirement for PABP. We also identified a novel ITAF, heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2), that stimulates SHMT1 IRES activity by binding to the 5'-UTR of the transcript and interacting with CUGBP1. Collectively, these data support a model for the IRES-mediated translation of SHMT1 whereby the circularization of the mRNA typically provided by the eukaryotic initiation factor (eIF) 4G/PABP/poly(A) tail interaction is achieved instead through the hnRNP H2/CUGBP1-mediated interaction of the 5'- and 3'-UTRs of the SHMT1 transcript. This circularization enhances the IRES activity of SHMT1 by facilitating the recruitment and/or recycling of ribosomal subunits, which bind to the transcript in the middle of the 5'-UTR and migrate to the initiation codon via eIF4A-mediated scanning.
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Affiliation(s)
- Jennifer T Fox
- Graduate Field of Biochemistry and Molecular and Cellular Biology, Cornell University, Ithaca, New York 14853, USA
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Abstract
BACKGROUND Islet transplantation success depends on the number and quality of islets transplanted. This study aimed at exploring the molecular mechanisms associated with cold pancreas preservation and their impact on islet cell survival and function. METHODS Rat pancreata were stored in cold University of Wisconsin preservation solution for short (3 hr; control) or long (18 hr) cold ischemia times (CIT). RESULTS Pancreata exposed to long CIT yielded lower islet numbers and showed reduced cellular viability; isolated islets displayed higher levels of phosphorylated stress-activated protein kinase (c-jun N-terminal Kinase and Mitogen-Activated Protein Kinase-p38), and chemokine (C-C) ligand-3, and lower levels of vascular endothelial growth factor, interleukins (IL)-9 and IL-10. Islets obtained from long-CIT pancreata were functionally impaired after transplantation. Differential proteomic expression in pancreatic tissue after CIT included increased eukaryotic translation elongation factor-1-alpha-1 (apoptosis related) and reduced Clade-B (serine protease inhibitor). CONCLUSIONS Our study indicates that cold ischemia stimulates inflammatory pathways (chemokine (c-c)ligand-3, phosphorylation of c-jun N-terminal Kinase and mitogen-activated protein kinase-p38, and eukaryotic translation elongation factor-1-alpha-1) and decreases repair/cytoprotective pathways (IL-10, vascular endothelial growth factor, and Clade-B), all of which may negatively affect the quality and mass of islets obtained from a donor pancreas.
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Fay J, Kelehan P, Lambkin H, Schwartz S. Increased expression of cellular RNA-binding proteins in HPV-induced neoplasia and cervical cancer. J Med Virol 2009; 81:897-907. [PMID: 19319956 DOI: 10.1002/jmv.21406] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The expression profile of a panel of RNA-binding proteins (heterogeneous ribonucleoprotein (hnRNP) A1, hnRNP C1/C2, hnRNP H, hnRNP I, ASF/SF2, SR proteins, HuR and U2AF(65)) and markers of differentiation, proliferation and neoplasia (cytokeratin (CK) 13, CK-14, proliferating cell nuclear antigen (PCNA), Syndecan-1 and p16INK4a) were analyzed in 50 formalin fixed paraffin embedded cervical tissues using immunohistochemistry. The samples included histologically normal cervical epithelium, human papillomavirus (HPV) induced low-grade and high-grade pre-malignant lesions and cervical cancers. All samples were tested for HPV DNA using nested PCR. Forty-nine of the 50 tissue samples tested positive for HPV, 27 tissue samples (54%) were HPV-16 positive and 4 samples (8%) were HPV-18 positive. The immunohistochemistry results detected different expression levels of the various proteins in basal epithelial cells in histologically normal epithelium followed by an increase in expression in the intermediate layers, whereas the superficial layers remained negative for all tested RNA-binding proteins. Expression of all RNA-binding proteins increased in neoplastic lesions and highest expression was detected in cervical cancers. p16INK4a had a stronger association with high-grade lesions when compared with the RNA-binding proteins. The expression profile of the RNA-binding proteins is similar to PCNA expression in histologically normal epithelium as well as in lesions (low-grade and high-grade) and cervical cancers. As PCNA expression has been suggested to mimic HPV E6/E7 expression in cervical epithelium, the results suggest the RNA-binding protein analyzed here regulate HPV early gene expression directly and late gene expression indirectly.
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Affiliation(s)
- Joanna Fay
- School of Biological Sciences, Dublin Institute of Technology, Dublin, Ireland
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47
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Hai Y, Cao W, Liu G, Hong SP, Elela SA, Klinck R, Chu J, Xie J. A G-tract element in apoptotic agents-induced alternative splicing. Nucleic Acids Res 2008; 36:3320-31. [PMID: 18440980 PMCID: PMC2425498 DOI: 10.1093/nar/gkn207] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Alternative splicing of a single pre-mRNA transcript can produce protein isoforms that promote either cell growth or death. Here we show that Ro-31-8220 (Ro), an apoptotic agent that inhibits protein kinase C and activates the c-Jun N terminal kinase, decreased the proportion of the cell growth-promoting Bcl-xL splice variant. Targeted mutagenesis analyses narrowed down a critical sequence to a 16-nt G-tract element (Gt16). Transferring this element to a heterologous gene conferred Ro response on an otherwise constitutive exon. The Ro effect was reduced by okadaic acid, an inhibitor of protein phosphatases PP1 and PP2A, in a concentration-dependent manner. Search in the human genome followed by RT–PCR identified a group of genes that contain similar exonic G-tract elements and are responsive to Ro. Moreover, the Gt16 element also mediates the regulation of alternative splicing by other cell apoptosis-inducers particularly retinoic acid. Therefore, the G-tract element likely plays a role in the apoptotic agents-induced alternative splicing of a group of genes. The functions of these genes imply that this regulation will have impact on cell growth/death.
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Affiliation(s)
- Yan Hai
- Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College. Kunming, China
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48
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Gruber-Olipitz M, Ströbel T, Chen WQ, Grotzer MA, Quehenberger F, Slavc I, Lubec G. Synthesis, chaperoning, and metabolism of proteins are regulated by NT-3/TrkC signaling in the medulloblastoma cell line DAOY. J Proteome Res 2008; 7:1932-44. [PMID: 18336001 DOI: 10.1021/pr700724a] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The human medulloblastoma cell line DAOY was transfected with Tropomyosin receptor kinase (TrkC), a marker for good prognostic outcome. Following TrkC-activation by its ligand neurotrophin-3, protein extracts from DAOY cells were run on 2DE with subsequent MALDI-TOF-TOF analysis and quantification in order to detect downstream effectors. Protein levels of translational, splicing, processing, chaperone, protein handling, and metabolism machineries were shown to depend on neurotrophin-3-induced TrkC activation probably representing pharmacological targets.
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49
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Skvortsov S, Skvortsova I, Stasyk T, Schiefermeier N, Neher A, Gunkel AR, Bonn GK, Huber LA, Lukas P, Pleiman CM, Zwierzina H. Antitumor activity of CTFB, a novel anticancer agent, is associated with the down-regulation of nuclear factor-kappaB expression and proteasome activation in head and neck squamous carcinoma cell lines. Mol Cancer Ther 2007; 6:1898-908. [PMID: 17575118 DOI: 10.1158/1535-7163.mct-06-0708] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
This study aimed to characterize the antitumor activity of 5-Chloro-N-[2-[2-(4-chloro-phenyl)-3-methyl-butoxy]-5-trifluoromethyl-phenyl]-2-hydroxy-benzamide (CTFB), a novel anticancer agent, in head and neck cancer cell lines, FaDu, SCC-25 and cisplatin-resistant CAL-27. CTFB was generated as a result of an extensive medicinal chemistry effort on a lead compound series discovered in a high-throughput screen for inducers of apoptosis. All cell lines showed significant growth delay in response to CTFB treatment at a concentration of 1 micromol/L with 17.16 +/- 2.08%, 10.92 +/- 1.22%, and 27.03 +/- 1.86% of cells surviving at 120 h in FaDu, CAL-27, and SCC-25, respectively. To define proteins involved in the mechanism of action of CTFB, we determined differences in the proteome profile of cell lines before and after treatment with CTFB using two-dimensional difference gel electrophoresis followed by computational image analysis and mass spectrometry. Eight proteins were found to be regulated by CTFB in all cell lines. All these proteins are involved in cytoskeleton formation and function and/or in cell cycle regulation. We showed that CTFB-induced cell growth delay was accompanied by cell cycle arrest at the G(0)-G(1) phase that was associated with the up-regulation of p21/WAF1 and p27/Kip1 expression and the down-regulation of cyclin D1. Furthermore, we showed that activity of CTFB depended on the down-regulation of nuclear factor-kappaB (NF-kappaB) and NF-kappaB p65 phosphorylated at Ser(536). The level of proteasome activity correlated with the response to CTFB treatment, and the down-regulation of NF-kappaB is accompanied by enhanced proteasome activity in all investigated head and neck cancer cell lines. In this report, we show that CTFB reveals multiple effects that lead to delayed cell growth. Our data suggest that this compound should be studied further in the treatment of head and neck cancer.
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Affiliation(s)
- Sergej Skvortsov
- Department of Internal Medicine, Innsbruck Medical University, Innsbruck, Austria.
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50
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Sun W, Xing B, Sun Y, Du X, Lu M, Hao C, Lu Z, Mi W, Wu S, Wei H, Gao X, Zhu Y, Jiang Y, Qian X, He F. Proteome analysis of hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein markers in hepatocellular carcinoma tissues. Mol Cell Proteomics 2007; 6:1798-808. [PMID: 17627933 DOI: 10.1074/mcp.m600449-mcp200] [Citation(s) in RCA: 194] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is a highly malignant tumor, and chronic infection with hepatitis B virus is one of its major risk factors. To identify the proteins involved in HCC carcinogenesis, we used two-dimensional fluorescence DIGE to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 12 hepatitis B virus-associated HCC patients were analyzed. A total of 61 spots were significantly up-regulated (ratio >/= 2, p </= 0.01) in tumor samples, whereas 158 spots were down-regulated (ratio </= -2, p </= 0.01). Seventy-one gene products were identified among these spots. Members of the heat shock protein 70 and 90 families were simultaneously up-regulated, whereas metabolism-associated proteins were decreased in HCC samples. The down-regulation of mitochondrial and peroxisomal proteins in these results suggested loss of special organelle functions during HCC carcinogenesis. Four metabolic enzymes involved in the methylation cycle in the liver were down-regulated in HCC tissues, indicating S-adenosylmethionine deficiency in HCC. Two gene products, glyceraldehyde-3-phosphate dehydrogenase and formimidoyltransferase-cyclodeaminase, were identified from inversely altered spots, suggesting that different isoforms or post-translational modifications of these two proteins might play different roles in HCC. For the first time, the overexpression of Hcp70/Hsp90-organizing protein and heterogeneous nuclear ribonucleoproteins C1/C2 in HCC tissues was confirmed by Western blot and then by immunohistochemistry staining in 70 HCC samples, suggesting their potential as protein tumor markers. In summary, we profiled proteome alterations in HCC tissues, and these results may provide useful insights for understanding the mechanism involved in the process of HCC carcinogenesis.
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MESH Headings
- Adult
- Amino Acid Sequence
- Ammonia-Lyases/metabolism
- Biomarkers, Tumor/analysis
- Blotting, Western
- Carcinoma, Hepatocellular/chemistry
- Carcinoma, Hepatocellular/enzymology
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/pathology
- Down-Regulation
- Electrophoresis, Gel, Two-Dimensional/methods
- Female
- Gene Expression Regulation, Neoplastic
- Heat-Shock Proteins/chemistry
- Heat-Shock Proteins/metabolism
- Heterogeneous-Nuclear Ribonucleoproteins/chemistry
- Heterogeneous-Nuclear Ribonucleoproteins/metabolism
- Humans
- Immunohistochemistry
- Male
- Middle Aged
- Molecular Sequence Data
- Neoplasm Proteins/analysis
- Neoplasm Proteins/chemistry
- Neoplasm Proteins/genetics
- Neoplasm Proteins/isolation & purification
- Proteome/analysis
- Reproducibility of Results
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- Subcellular Fractions
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Affiliation(s)
- Wei Sun
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, P.R. China
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