Deubiquitinases (DUBs) are increasingly linked to the regulation of fundamental processes in normal and cancer cells, including DNA replication and repair, programmed cell death, and oncogenes and tumor suppressors signaling. Here evidence is presented that the deubiquitinase OTUD6B regulates protein synthesis in non-small cell lung cancer (NSCLC) cells, operating downstream from mTORC1. OTUD6B associates with the protein synthesis initiation complex and modifies components of the 48S preinitiation complex. The two main OTUD6B splicing isoforms seem to regulate protein synthesis in opposing fashions: the long OTUD6B-1 isoform is inhibitory, while the short OTUD6B-2 isoform stimulates protein synthesis. These properties affect NSCLC cell proliferation, since OTUD6B-1 represses DNA synthesis while OTUD6B-2 promotes it. Mutational analysis and downstream mediators suggest that the two OTUD6B isoforms modify different cellular targets. OTUD6B-2 influences the expression of cyclin D1 by promoting its translation while regulating (directly or indirectly) c-Myc protein stability. This phenomenon appears to have clinical relevance as NSCLC cells and human tumor specimens have a reduced OTUD6B-1/OTUD6B-2 mRNA ratio compared to normal samples. The global OTUD6B expression level does not change significantly between non-neoplastic and malignant tissues, suggesting that modifications of splicing factors during the process of transformation are responsible for this isoform switch. Implications Because protein synthesis inhibition is a viable treatment strategy for NSCLC, these data indicate that OTUD6B isoform 2, being specifically linked to NSCLC growth, represents an attractive, novel therapeutic target and potential biomarker for early diagnosis of malignant NSCLC.
<div>Abstract<p>Deubiquitinases (DUB) are increasingly linked to the regulation of fundamental processes in normal and cancer cells, including DNA replication and repair, programmed cell death, and oncogenes and tumor suppressor signaling. Here, evidence is presented that the deubiquitinase OTUD6B regulates protein synthesis in non–small cell lung cancer (NSCLC) cells, operating downstream from mTORC1. OTUD6B associates with the protein synthesis initiation complex and modifies components of the 48S preinitiation complex. The two main OTUD6B splicing isoforms seem to regulate protein synthesis in opposing fashions: the long OTUD6B-1 isoform is inhibitory, while the short OTUD6B-2 isoform stimulates protein synthesis. These properties affect NSCLC cell proliferation, because OTUD6B-1 represses DNA synthesis while OTUD6B-2 promotes it. Mutational analysis and downstream mediators suggest that the two OTUD6B isoforms modify different cellular targets. OTUD6B-2 influences the expression of cyclin D1 by promoting its translation while regulating (directly or indirectly) c-Myc protein stability. This phenomenon appears to have clinical relevance as NSCLC cells and human tumor specimens have a reduced OTUD6B-1/OTUD6B-2 mRNA ratio compared with normal samples. The global OTUD6B expression level does not change significantly between nonneoplastic and malignant tissues, suggesting that modifications of splicing factors during the process of transformation are responsible for this isoform switch.</p><p><b>Implications:</b> Because protein synthesis inhibition is a viable treatment strategy for NSCLC, these data indicate that OTUD6B isoform 2, being specifically linked to NSCLC growth, represents an attractive, novel therapeutic target and potential biomarker for early diagnosis of malignant NSCLC. <i>Mol Cancer Res; 15(2); 117–27. ©2016 AACR</i>.</p></div>
<p>Table ST1. List of oligonucleotides and siRNAs used in this study. Table ST2. List of antibodies used in this study. Figure S1. Sequence alignment of OTUD6A and OTUD6B (isoform 1) performed using Clustal omega software. Figure S2. RNA silencing to OTUD2 and OTUD5 in NSCLC cells does not affect the protein synthesis rate. Figure S3. Comassie blue staining of a representative SDS PAGE gel loaded with 40 μg of proteins obtained from total cell lysate (1), nuclear fraction (2), postmitochondrial fraction (3), postribosomal fraction (4) and ribosomes (5). Note the typical enrichment in low molecular weigh bands (ribosomal proteins) of the ribosomal fraction. Figure S4. OTUD6B coimmunoprecipitates with eIF4G and other eIF4F components. Figure S5. Schematic (Top) and sequence alignment (Bottom) of OTUD6B isoforms. Large spiral: coiled coil domain. Figure S6. Evidence of allelic mutation (arrow) of the OTUD6B catalytic core in HCC827. Figure S7. OTUD6B-1 and OTUD6B-2 cause cell death when overexpressed in NSCLC cells. Figure S8. Hematoxylin/Eosin staining of 8μm-thick, frozen slides of representative normal lung.</p>
<div>Abstract<p>Deubiquitinases (DUB) are increasingly linked to the regulation of fundamental processes in normal and cancer cells, including DNA replication and repair, programmed cell death, and oncogenes and tumor suppressor signaling. Here, evidence is presented that the deubiquitinase OTUD6B regulates protein synthesis in non–small cell lung cancer (NSCLC) cells, operating downstream from mTORC1. OTUD6B associates with the protein synthesis initiation complex and modifies components of the 48S preinitiation complex. The two main OTUD6B splicing isoforms seem to regulate protein synthesis in opposing fashions: the long OTUD6B-1 isoform is inhibitory, while the short OTUD6B-2 isoform stimulates protein synthesis. These properties affect NSCLC cell proliferation, because OTUD6B-1 represses DNA synthesis while OTUD6B-2 promotes it. Mutational analysis and downstream mediators suggest that the two OTUD6B isoforms modify different cellular targets. OTUD6B-2 influences the expression of cyclin D1 by promoting its translation while regulating (directly or indirectly) c-Myc protein stability. This phenomenon appears to have clinical relevance as NSCLC cells and human tumor specimens have a reduced OTUD6B-1/OTUD6B-2 mRNA ratio compared with normal samples. The global OTUD6B expression level does not change significantly between nonneoplastic and malignant tissues, suggesting that modifications of splicing factors during the process of transformation are responsible for this isoform switch.</p><p><b>Implications:</b> Because protein synthesis inhibition is a viable treatment strategy for NSCLC, these data indicate that OTUD6B isoform 2, being specifically linked to NSCLC growth, represents an attractive, novel therapeutic target and potential biomarker for early diagnosis of malignant NSCLC. <i>Mol Cancer Res; 15(2); 117–27. ©2016 AACR</i>.</p></div>
<p>Table ST1. List of oligonucleotides and siRNAs used in this study. Table ST2. List of antibodies used in this study. Figure S1. Sequence alignment of OTUD6A and OTUD6B (isoform 1) performed using Clustal omega software. Figure S2. RNA silencing to OTUD2 and OTUD5 in NSCLC cells does not affect the protein synthesis rate. Figure S3. Comassie blue staining of a representative SDS PAGE gel loaded with 40 μg of proteins obtained from total cell lysate (1), nuclear fraction (2), postmitochondrial fraction (3), postribosomal fraction (4) and ribosomes (5). Note the typical enrichment in low molecular weigh bands (ribosomal proteins) of the ribosomal fraction. Figure S4. OTUD6B coimmunoprecipitates with eIF4G and other eIF4F components. Figure S5. Schematic (Top) and sequence alignment (Bottom) of OTUD6B isoforms. Large spiral: coiled coil domain. Figure S6. Evidence of allelic mutation (arrow) of the OTUD6B catalytic core in HCC827. Figure S7. OTUD6B-1 and OTUD6B-2 cause cell death when overexpressed in NSCLC cells. Figure S8. Hematoxylin/Eosin staining of 8μm-thick, frozen slides of representative normal lung.</p>
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