miR-490 suppresses telomere maintenance program and associated hallmarks in glioblastoma
Authors: Maria A Blasco
Kurt Whittemore
Deependra Kushwah
Omkar Suhas Vinchure
Ritu Kulshreshtha
Journal: Cellular and Molecular Life Sciences (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: DNA damage; Glioblastoma; REST; TERF2; Telomeres

Abstract:  Glioblastoma (GBM) is the most aggressive cancer of central nervous system with worst patient outcome. Telomere maintenance is a crucial mechanism governing GBM initiation and progression making it an attractive target. microRNAs (miRNAs) have shown therapeutic potential in GBM. Earlier, we showed miR-490 is downregulated in GBM patients and plays a tumor suppressive role. Here, we show that miR-490 regulates telomere maintenance program in GBM by directly targeting Telomeric Repeat-binding Factor 2 (TERF2) of the shelterin complex, Tankyrase 2 (TNKS2) and Serine/Threonine-protein kinase, SMG1. Overexpression of miR-490 resulted in effects characteristic to hampered telomere maintenance via TERF2 inhibition. These include induction of telomere dysfunction-induced foci and global DNA damage (53BP1 foci), along with an increase in p-γH2AX levels. Further, it led to inhibition of telomere maintenance hallmarks via reduced stemness (SOX2 and SOX4 downregulation) and induction of senescence (H3K9me3 marks gain and SIRT1 downregulation). It also initiated downstream DNA damage response (DDR) leading to p53 pathway activation. Moreover, microarray data analysis highlighted an overlap between miR-490 expression and REST-inhibition responses in GBM. Thus, miR-490-mediated targeting of telomere maintenance could be therapeutically important in GBM. 

Retracted: No

PubPeer comments:
  Six images of the metaphase spreads (random) per slide were obtained using a confocal microscope (Leica TCSSP5) followed by quantification of the telomere probe fluorescent signal. - authors write in the materials and methods that they have analysed 6 images for telomere dysfunction analysis on the metaphases. On what basis this number was chosen. Out of millions of cells, only 6 were analysed?  Quantification of Q-FISH data represented as average telomere spot fluorescence. Number of spots analyzed for each sample is as follows: U87MG PCDNA (4975 spots), U87MG PC-490 (8257 spots), T98G PCDNA (19,247 spots) and T98G PC-490 (14,812 spots) - this clearly shows the lack of knowledge on chromosome status in glioblastoma cell lines. Both these cell lines display not diploid cells and they show dynamic changes in the karyotype. The authors should have indicated average number of chromosomes in each cell type. Number of telomere spots is misleading as the number of chromosomes in each cell may change in a pool of cells. Moreover, they have analysed only 6 metaphase chromosomes. When you grow cancer cells in culture, they display dynamic changes in telomere length as well status over time. TO achieve statistical significance, analysis of 6 metaphases may not be sufficient. Expert statisticians may comment here. How the telomere fluorescence was measured? how did they come up with that arbitrary unit? There was no reference indicated what was the internal control for the fluorescence analysis? It appears that the authors selectively avoided referring to some of the recent studies on telomerase inhibition in glioblastoma cells. I will list down other issues with the study in the next comment (Hope this message doesn t get moderated). , 
 This paper has used two glioblastoma cell lines - U87MG and T98G both are cancer cell lines and contain aneuploid cells - mostly additional chromosomes. One of the quantifications of telomere dysfunction - number of telomere spots on chromosome ends (Figures 2 a and b). When spontaneously these cell lines have altered chromosome number which may vary upon continuous passaging, counting number telomere spots may not be a good parameter to use to determine telomere dysfunction. Variation in the number of telomere spots may not mean much - as the cancer cell lines are believed to be undergoing BFB cycles. Please clarify. Second point: Number of metaphases used in the telomere length quantification in QFISH. They have used 6 metaphases out of millions of cells and compared to other assays. This is not valid for any statistical comparison. Here again, if the average number of chromosomes vary significantly among the metaphase chosen, QFISH values may be erroneous. The number of metaphase analysed is too low for any scientific validation. A detailed description of QFISH is not given or not referred to any previous papers. How did they come up with ~30 Telomere spot fluorescence (a.u.)? (Fig 2 b) , 
 We thank Psychotria iteophylla for their questions regarding our article. Please find below the response to your concerns from our side. First point: u2018One of the quantifications of telomere dysfunction - number of telomere spots on chromosome ends (Figures 2 a and b).u2019 The number of telomere spots on chromosome ends was not used as a quantification of telomere dysfunction as pointed out. The figures 2a and 2b represent the quantification of the average fluorescent signal of telomere spots (corresponding to average telomere length) as analyzed by Q-FISH. Second point: u2018Number of metaphases used in the telomere length quantification in QFISH.u2019 We had acquired six images of metaphase spreads per slide of each sample. That corresponds to = 18 metaphases per sample. The corresponding number of spots analyzed per sample for quantification is as follows: U87MG PCDNA (4975 spots), U87MG PC-490 (8257 spots), T98G PCDNA (19,247 spots) and T98G PC-490 (14,812 spots) which is valid for statistical comparison. As rightfully pointed out by Psychotria Iteophylla, the number of chromosomes in cancer cell lines could vary over time and passaging. We had used the same cells around the same passage number and directly compared two different treatment groups. Also, here too the average fluorescent intensities of telomere spots are quantified for analysis instead of the actual chromosome number. u2018How did they come up with ~30 Telomere spot fluorescence (a.u.)? (Fig 2 b)u2019 The telomere spot fluorescence is computed as arbitrary units of fluorescence by the image processing software. Thank you. ,