miR-380-5p-mediated repression of TEP1 and TSPYL5 interferes with telomerase activity and favours the emergence of an "ALT-like" phenotype in diffuse malignant peritoneal mesothelioma cells

Our plain-English summary of miR-380-5p-mediated repression of TEP1 and TSPYL5 interferes with telomerase activity and favours the emergence of an "ALT-like" phenotype in diffuse malignant peritoneal mesothelioma cells, by G. Cimino-Reale, P. Gandellini, F. Santambrogio, et al (Journal of Hematology & Oncology, 2017).

The study

• Authors: G. Cimino-Reale, P. Gandellini, F. Santambrogio, M. Recagni, N. Zaffaroni, M. Folini
• Journal: Journal of Hematology & Oncology
• Published: 2017;10(1):140
• Cited by: 29 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Understanding the molecular/cellular underpinnings of diffuse malignant peritoneal mesothelioma (DMPM), a fatal malignancy with limited therapeutic options, is of utmost importance for the fruitful management of the disease. In this context, we previously found that telomerase activity (TA), which accounts for the limitless proliferative potential of cancer cells, is prognostic for disease relapse and cancer-related death in DMPM patients. Consequently, the identification of factors involved in telomerase activation/regulation may pave the way towards the development of novel therapeutic interventions for the disease. Here, the capability of miR-380-5p, a microRNA negligibly expressed in telomerase-positive DMPM clinical specimens, to interfere with telomerase-mediated telomere maintenance and, hence, with cancer cell growth was assessed on preclinical models of DMPM.

Methods: DMPM cells were transfected with a miR-380-5p synthetic precursor, and the effects of miRNA replacement were evaluated in terms of growing capability, induction of apoptosis and interference with TA. Reiterated weekly transfections were also performed in order to analyse the phenotype arising upon prolonged miR-380-5p reconstitution in DMPM cells.

Results: The ectopic expression of miR-380-5p elicited a remarkable inhibition of TA and resulted in DMPM cell growth impairment and apoptosis induction. In particular, we demonstrated for the first time that these effects were the result of a molecular circuitry converging on telomerase associated protein 1 (TEP1), where the miRNA was able to target the gene both directly in unconventional targeting modality and indirectly via p53 accumulation consequent to miRNA-mediated downregulation of testis-specific protein, Y-encoded-like 5 gene. Moreover, miR-380-5p did not cause telomere attrition and cell growth arrest in long-term DMPM transfectants, which in turn showed slightly elongated telomeres and molecular features (e.g. c-circle DNA and reduced expression levels of chromatin remodeler ATRX) resembling an alternative lengthening of telomeres (ALT) phenotype.

Conclusions: miR-380-5p interferes with TA in DMPM cells by targeting TEP1. Notably, in the long-term setting, miR-380-5p-mediated impairment of TA did not result in telomere attrition. Instead, a phenotype reminiscent of ALT emerged in DMPM cells as possible compensatory pathway that safeguards DMPM cell growth, an event that may be regarded as a potential resistance mechanism to anticancer therapies based on telomerase inhibitors.

In plain English

This was a laboratory study in cultured cell lines, and no patients were treated. The authors also reported that miR-380-5p was significantly under-expressed in telomerase-positive diffuse malignant peritoneal mesothelioma (DMPM) tissue specimens compared with telomerase-negative DMPM or normal peritoneal specimens. They transfected three telomerase-positive DMPM cell lines (STO, MP8, MP4) with a synthetic miR-380-5p precursor, alongside A549 lung cancer, ALT-positive U-2 Os and normal mesothelial MES-F cells. Short-term miR-380-5p expression mildly but significantly slowed DMPM cell growth and inhibited telomerase activity (61 ± 1.7%, P < 0.01). It was accompanied by p53 and p21waf1 accumulation and apoptosis markers, and it did not impair growth of normal mesothelial or telomerase-negative cells. TEP1 fell in all three DMPM lines but not in U-2 Os, and TEP1 knockdown in STO cells reproduced the telomerase inhibition and growth impairment. Experiments indicated TEP1 and TSPYL5 as direct targets. In STO cells given weekly transfections for 3 months, telomeres did not shorten (mean length 3.29 ± 0.15 to 4.28 ± 0.30 Kb, P < 0.005), ATRX expression fell and C-circle DNA appeared. The authors concluded that an "ALT-like" phenotype emerged, possibly as a compensatory pathway.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) experimental study in human cancer cell lines, with short-term and repeated long-term transfection of a microRNA precursor, plus expression analysis of miR-380-5p in DMPM and normal peritoneal tissue specimens
• Who was studied: Human cell lines: diffuse malignant peritoneal mesothelioma lines STO, MP8 and MP4; A549 lung cancer cells; U-2 Os osteosarcoma cells (telomerase-negative, ALT-positive); normal adult mesothelial MES-F cells; HeLa used as a control in the C-circle assay. Also telomerase-positive and telomerase-negative DMPM tissue specimens and normal peritoneal specimens (numbers not given in the text). The laboratory was in Milan, Italy, and the article was received in March 2017.
• Exposure: Not an asbestos or occupational exposure study. Cells were exposed to transfected oligonucleotides: miR-380-5p synthetic precursor (20 nM) or negative control, the miRNA inhibitor LNA380 (100 nM), siRNAs against TEP1, TSPYL5 and p53 (50 nM), and target protector oligomers (100 nM). Some experiments also used doxorubicin at 3 micrograms per ml.
• Main results:
  • miR-380-5p was significantly under-expressed in telomerase-positive DMPM tissue specimens compared with telomerase-negative DMPM specimens or normal peritoneal specimens
  • In DMPM cells, miR-380-5p expression slowed growth and inhibited telomerase activity by 61 ± 1.7% (P less than 0.01)
  • The same transfection did not impair growth of normal mesothelial cells or telomerase-negative cancer cells
  • miR-380-5p transfection was accompanied by accumulation of p53 and p21waf1 and by apoptosis markers in DMPM cells
  • TEP1 mRNA and protein decreased in all three telomerase-positive DMPM lines but not in telomerase-negative U-2 Os cells
  • siRNA knockdown of TEP1 in STO cells reproduced the telomerase inhibition and growth impairment seen with miR-380-5p
  • Reporter gene assays indicated that the TSPYL5 3'-UTR is directly targeted by miR-380-5p (P less than 0.01, wild-type versus deleted construct)
  • Depletion of TSPYL5 in STO and A549 cells led to p53 accumulation, reduced TEP1 protein and telomerase inhibition
  • After 3 months of repeated transfection, STO cells did not lose telomere length. Mean telomere length in miR-380-5p STO cells went from 3.29 ± 0.15 Kb at week 1 to 4.28 ± 0.30 Kb at month 3 (P less than 0.005). In control (preNeg) STO cells it went from 3.35 ± 0.17 to 3.50 ± 0.20 Kb (not significant).
  • Long-term miR-380-5p STO transfectants showed reduced ATRX expression and detectable C-circle DNA, and they continued growing at a reduced rate without growth arrest
• The authors' conclusion: The authors concluded that miR-380-5p interfered with telomerase activity and caused growth impairment and apoptosis in DMPM cell models. They also concluded that long-term miR-380-5p expression led to cells with features partly reminiscent of an "ALT-like" phenotype, which could act as a compensatory mechanism sustaining cell growth.
• Limitations the authors state:
  • The authors state that the hypothesis that ALT may emerge in telomerase-positive tumours after prolonged telomerase inhibition has not been confirmed in the clinical setting
  • The authors describe the long-term phenotype only as partly resembling ALT, which they term "ALT-like"
  • The authors state that genome-wide analysis is still needed to identify the factors responsible for the "ALT-like" phenotype and to validate them as targets

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Peritoneal Mesothelioma, Apoptosis.

Related studies

• SV40 infection induces telomerase activity in human mesothelial cells — R. Foddis, A. De Rienzo, et al. (2002)
• EphrinA1 inhibits malignant mesothelioma tumor growth via let-7 microRNA-mediated repression of the RAS oncogene — N. Khodayari, K.A. Mohammed, et al. (2011)
• Telomerase activity in human pleural mesothelioma — K. Dhaene, R. Hübner, et al. (1998)
• Telomerase activity in pleural malignant mesotheliomas — A.Y. Au, T. Hackl, et al. (2011)
• Telomerase activity analyzed with TRAP in situ provides additional information in effusions remaining equivocal after immunocytochemistry and hyaluronan analysis — E. Adell, A. Dejmek (2014)
• miR-550a-3p is a prognostic biomarker and exerts tumor-suppressive functions by targeting HSP90AA1 in diffuse malignant peritoneal mesothelioma — R. El Bezawy, S. Percio, et al. (2022)

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