Telomerase variant A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal carcinomas
Authors: Thomas Ried
Mahadev Rao
Bogdan Dumitriu
Curtis C Harris
Tricia F Kunst
Hesed M Padilla-Nash
Scott Atay
Suzanne Inchauste
Nathan R Peterson
Alan K Meeker
Neal S Young
Mary Zhang
David G Beer
Danny Wangsa
Susan H Garfield
David S Schrump
Yuwei Zhang
Rodrigo Calado
Julie A Hong
Peter J McCormick
Sichuan Xi
Nasser K Altorki
Sandra Burkett
Alan G Casson
Journal: PLoS ONE (2014)
Discipline: Multidisciplinary
Keywords: telomerase mutations; esophageal carcinomas; TERT variants; telomere length; chromosomal stability
Affiliations: Thoracic Surgery Section, Thoracic and GI Oncology Branch; National Cancer Institute, Bethesda, Maryland, United States of America., National Heart, Lung, and Blood Institute, Bethesda, Maryland, United States of America., Departments of Pathology and Oncology, Johns Hopkins University of Medicine, Baltimore, Maryland, United States of America., Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland, United States of America., Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, Maryland, United States of America., Section of Cancer Genomics, National Cancer Institute, Bethesda, Maryland, United States of America., Comparative Molecular Cytogenetics Core Facility, National Cancer Institute, Frederick, Maryland, United States of America., Department of Thoracic Surgery, Weill Cornell Medical Center, New York, New York, United States of America., Department of Surgery, University of Saskatchewan, Saskatoon, Saskatchewan, Canada., Section of Thoracic Surgery, University of Michigan Medical Center, Ann Arbor, Michigan, United States of America., Laboratory of Human Carcinogenesis, National Cancer Institute, Bethesda, Maryland, United States of America.

Abstract:  Background: Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas. Methods: Sequencing techniques were used to evaluate mutational status of telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and β-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability. Results: Sequencing analysis revealed one deletion involving TERC (TERC del 341-360), and two non-synonymous TERT variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p&lt;0.01). Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-β-catenin complex, markedly depleted β-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin™ exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage. Conclusions: A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal cancer cells. These findings warrant further analysis of A279T expression in esophageal cancers and premalignant esophageal lesions. 

Retracted: No

PubPeer comments:
 This is one of several papers from different author teams that report using the same forward and reverse primers for quantitative PCR of miR-296-5p or miR-296-3p. Here, qRT-PCR analysis is said to have been done with miR-296-5p specific primers (Forward, 5u02b9-TGCCTAATTCAGAGGGTTGG-3u02b9; Reverse, 5u02b9-CTCCACTCCTGGCACACAG-3u02b9) . Unfortunately, no reliable results for miR-296-5p would have been obtained with these primers. Here is the precursor sequence for miR-296, with the miR-296-5p arm (the one that is supposed to be amplified in this paper) in bold: AGGACCCUUCCAG**AGGGCCCCCCCUCAAUCCUGU**UGUGCCUAAUUCAGAGGGUUGGGUGGAGGCUCUCCUGAAGGGCUCU And here in bold is where the published  forward primer sequence would bind: AGGACCCUUCCAGAGGGCCCCCCCUCAAUCCUGUUG**UGCCUAAUUCAGAGGGUUGG**GUGGAGGCUCUCCUGAAGGGCUCU Obviously, this primer does not capture mature miR-296-5p at all. Also, the reverse primer recognizes a sequence adjacent to the miR-296 precursor in the genome, but this is not part of the precursor RNA. These forward and reverse primers might amplify genomic DNA, or possibly a region from a longer primary transcript. They could not tell us about the mature -5p miRNA sequence that is the putative subject of this study. , 
 The papers that use these primers and methods incorrectly include the following: 1. [Zhang et al, 2019] 2. [Huang et al, 2021] 3. [Fan et al, 2020] 4. [Wang et al, 2020] 5. [Wang et al, 2020] 6. [Li et al, 2019] 7. [Zhang et al, 2021] 8. [Li et al, 2017] 9. [Ni et al, 2020] It appears to me that the first use of these primers in the literature was by a group that used them correctly, to examine the levels of the **primary** miR-296-containing transcript (Lee, et al, Mol Cells, 2017, and I want to stress that I find no problems with this paper.) It would seem that the methods and primers were subsequently copied without an understanding of the difference between primary, precursor, and mature miRNAs, or of the fact that precursor can give rise to different mature miRNAs. It is sobering for me to realize that entire author teams could be involved in putative microRNA research without understanding the fundamentals. , 
 #2  entire author teams could be involved in putative microRNA research without understanding the fundamentals It is conceivable that many of these author teams are **not** involved in microRNA research, or indeed in any research at all, but they needed a paper for career purposes. , 
 #1 Dear Insana, Thank you for your concern. When we were writing the article, we mistakenly copied the previous primer information, resulting in the writing error of miRNA-296-5p primer. The correct primers should be 5 -AGGGCCCCCCCTCAA-3 (F) and 5 -CAGTGCAGGGTCCGAGGTAT-3 (R). I apologize for the unintentional mistake. Many thanks for your kind reminding, we are very sorry for the error of the primers. And we have corrected this and we have send a correction to the journal and expect to publish Erratum soon. We will be more rigorous in the future scientific research work. Thank you for your interest in our work! Sincerely, Shi. ,