Identification of cis- and trans-acting elements regulating calretinin expression in mesothelioma cells

Our plain-English summary of Identification of cis- and trans-acting elements regulating calretinin expression in mesothelioma cells, by J. Kresoja-Rakic, E. Kapaklikaya, G. Ziltener, et al (Oncotarget, 2016).

The study

• Authors: J. Kresoja-Rakic, E. Kapaklikaya, G. Ziltener, D. Dalcher, R. Santoro, B.C. Christensen, K.C. Johnson, B. Schwaller, W. Weder, R.A. Stahel, E. Felley-Bosco
• Journal: Oncotarget
• Published: 2016;7(16):21272-86
• Cited by: 15 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Calretinin (CALB2) is a diagnostic marker for epithelioid mesothelioma. It is also a prognostic marker since patients with tumors expressing high calretinin levels have better overall survival. Silencing of calretinin decreases viability of epithelioid mesothelioma cells. Our aim was to elucidate mechanisms regulating calretinin expression in mesothelioma. Analysis of calretinin transcript and protein suggested a control at the mRNA level. Treatment with 5-aza-2'-deoxycytidine and analysis of TCGA data indicated that promoter methylation is not likely to be involved. Therefore, we investigated CALB2 promoter by analyzing ~1kb of genomic sequence surrounding the transcription start site (TSS) + 1 using promoter reporter assay. Deletion analysis of CALB2 proximal promoter showed that sequence spanning the -161/+80bp region sustained transcriptional activity. Site-directed analysis identified important cis-regulatory elements within this -161/+80bp CALB2 promoter. EMSA and ChIP assays confirmed binding of NRF-1 and E2F2 to the CALB2 promoter and siRNA knockdown of NRF-1 led to decreased expression of calretinin. Cell synchronization experiment showed that calretinin expression was cell cycle regulated with a peak of expression at G1/S phase. This study provides the first insight in the regulation of CALB2 expression in mesothelioma cells.

In plain English

This laboratory study examined what controls calretinin, a protein used to diagnose epithelioid mesothelioma, in mesothelioma cells. It did not study asbestos exposure or disease risk in people. The authors used 11 mesothelioma cell lines, an immortalized mesothelial line and HEK293 cells, plus Cancer Genome Atlas data on 87 tumors; methylation comparisons used the 57 epithelioid and 23 biphasic tumors. Calretinin gene activity was significantly higher in epithelioid cell lines and tracked protein levels. A drug that removes DNA methylation did not raise calretinin; protein levels fell instead, especially in SPC111 cells. In tumors, methylation of the gene's control region (promoter) was generally low. The authors judged methylation unlikely to explain tumor-type differences. A short promoter section, from 161 base pairs before to 80 after the gene's start site, sufficed to drive activity. Mutating a site recognized by the proteins NRF-1 and E2F2 cut that activity by 80%. Both proteins bound the promoter, and silencing NRF-1 lowered calretinin. Calretinin peaked at the G1/S stage of the cell cycle. The authors concluded that NRF-1 and E2F2 activate calretinin expression, which depends on the cell cycle.

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) study using human cell lines, combined with analysis of publicly available tumor data from The Cancer Genome Atlas (TCGA)
• Who was studied: 11 mesothelioma cell lines (5 epithelioid, 4 biphasic, 2 sarcomatoid), one SV-40 immortalized human pleural mesothelial cell line (MeT5A) and HEK293 cells. TCGA data were available for 87 mesotheliomas: 57 epithelioid, 23 biphasic, 5 diffuse not otherwise specified and 2 sarcomatoid. The methylation comparisons used the 57 epithelioid and 23 biphasic tumors.
• Exposure: No asbestos or other fiber exposure was studied. Laboratory treatments included:
• the hypomethylating drug 5-aza-2'-deoxycytidine, at 100 and 250 nM for seven days in ZL55 and SPC111 cells
• promoter reporter constructs with deletions and mutations
• silencing of NRF-1
• cell-cycle synchronization
• Main results:
  • Across the cell line panel, calretinin gene (CALB2) transcript levels were significantly higher in the epithelioid type (p = 0.0285). CALB2 mRNA was strongly positively correlated with calretinin protein levels (p = 0.0002).
  • In ZL55 and SPC111 cells, 5-aza-2'-deoxycytidine strongly raised the control genes CTAG1B and MAGE-C1. It did not increase calretinin mRNA or protein; calretinin protein decreased, especially in SPC111 cells.
  • In TCGA tumors (57 epithelioid, 23 biphasic), CALB2 promoter methylation was generally low. It was not significantly negatively correlated with CALB2 gene expression in either epithelioid or biphasic tumors.
  • The shortest promoter fragment, from 161 base pairs upstream to 80 base pairs downstream of the start site, was as active as longer fragments in all tested cell lines. Its activity was correlated with CALB2 expression (p = 0.017).
  • In ZL55 and SPC111 cells, all mutated promoter constructs had significantly lower activity than the control construct. Mutating the E2F2/NRF-1-like site at position minus 64 reduced activity by 80% compared with the unmutated promoter.
  • In ZL55 cells, chromatin immunoprecipitation showed NRF-1 significantly enriched at the CALB2 promoter compared with the CDC25A promoter, and E2F2 bound both promoters. Silencing NRF-1 lowered calretinin in SPC111 cells.
  • In synchronized ZL55 and SPC111 cells, calretinin was high at the G1/S transition. It decreased significantly 5 hours after thymidine removal, when most cells were in G2/M.
• The authors' conclusion: The authors concluded that NRF-1 and E2F2 activate calretinin expression at the transcriptional level and that calretinin expression levels are cell cycle-dependent. They also concluded that promoter methylation is not driving the differences in calretinin expression between mesothelioma tumor types.
• Limitations the authors state:
  • The authors state that the work cannot directly exclude post-transcriptional mechanisms, such as RNA stability, in regulating calretinin expression.
  • The authors note that the control plasmid used to normalize reporter results might in theory be affected by differing Sp1 levels between cell lines. They reasoned that this did not explain the roughly 30-fold differences they observed.

Topics

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

Medical terms in this study, explained in our Lexicon: Calretinin, Mesothelioma, Epithelioid, Overall Survival.

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• Identification of a differential expression of two cDNAs between malignant mesothelioma and normal mesothelial cells using the RNA fingerprint method — S. Frank, B.U. von Specht, et al. (1998)
• Expression of Calretinin, Marker of Mesothelial Differentiation, in Pancreatic Ductal Adenocarcinoma: A Potential Diagnostic Pitfall — G. Askan, O. Basturk (2021)

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