Regulation of calretinin in malignant mesothelioma is mediated by septin 7 binding to the CALB2 promoter

Our plain-English summary of Regulation of calretinin in malignant mesothelioma is mediated by septin 7 binding to the CALB2 promoter, by W. Blum, L. Pecze, J.W. Rodriguez, et al (BMC Cancer, 2018).

The study

• Authors: W. Blum, L. Pecze, J.W. Rodriguez, M. Steinauer, B. Schwaller
• Journal: BMC Cancer
• Published: 2018;18(1):475
• Cited by: 33 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: The calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). MM is a very aggressive tumor strongly linked to asbestos exposure and with no existing cure so far. The mechanisms of calretinin regulation, as well as its distinct function in MM are still poorly understood.

Methods: We searched for transcription factors binding to the CALB2 promoter and modulating calretinin expression. For this, DNA-binding assays followed by peptide shotgun-mass spectroscopy analyses were used. CALB2 promoter activity was assessed by dual-luciferase reporter assays. Furthermore, we analyzed the effects of CALB2 promoter-binding proteins by lentiviral-mediated overexpression or down-regulation of identified proteins in MM cells. The modulation of expression of such proteins by butyrate was determined by subsequent Western blot analysis. Immunohistochemical analysis of embryonic mouse lung tissue served to verify the simultaneous co-expression of calretinin and proteins interacting with the CALB2 promoter during early development. Finally, direct interactions of calretinin with target proteins were evidenced by co-immunoprecipitation experiments.

Results: Septin 7 was identified as a butyrate-dependent transcription factor binding to a CALB2 promoter region containing butyrate-responsive elements (BRE) resulting in decreased calretinin expression. Accordingly, septin 7 overexpression decreased calretinin expression levels in MM cells. The regulation was found to operate bi-directionally, i.e. calretinin overexpression also decreased septin 7 levels. During murine embryonic development calretinin and septin 7 were found to be co-expressed in embryonic mesenchyme and undifferentiated mesothelial cells. In MM cells, calretinin and septin 7 colocalized during cytokinesis in distinct regions of the cleavage furrow and in the midbody region of mitotic cells. Co-immunoprecipitation experiments revealed this co-localization to be the result of a direct interaction between calretinin and septin 7.

Conclusions: Our results demonstrate septin 7 not only serving as a "cytoskeletal" protein, but also as a transcription factor repressing calretinin expression. The negative regulation of calretinin by septin 7 and vice versa sheds new light on mechanisms possibly implicated in MM formation and identifies these proteins as transcriptional regulators and putative targets for MM therapy.

In plain English

This was a laboratory study using human cell lines and mouse tissue. It did not study asbestos exposure or patients. The authors examined how cells control production of calretinin, a protein used to diagnose malignant mesothelioma. They treated mesothelioma cell lines and immortalized mesothelial cells with butyrate, a fatty acid made by bacteria. Calretinin levels rose with the butyrate dose in all cell lines tested. A stretch of the calretinin gene's control region (BRE9–13) raised reporter activity after butyrate treatment, to 200 ± 24% of control in MSTO-211H cells and 123 ± 18% in ZL55 cells. Less septin 7 was bound to this region after butyrate treatment. Adding extra septin 7 to MSTO-211H cells lowered calretinin, and adding extra calretinin lowered septin 7. The two proteins were found together in mouse embryo tissue. Septin 7 was pulled down with calretinin from mesothelioma cells engineered to make extra calretinin. The authors concluded that septin 7 represses calretinin. They suggested that targeting septin 7 might be a possible therapy strategy.

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 of human cell lines, with immunohistochemistry on mouse embryo tissue and mouse primary mesothelial cells; DNA-binding assays, mass spectroscopy, luciferase reporter assays, lentiviral overexpression/knock-down, Western blots, co-immunoprecipitation
• Who was studied: Human mesothelioma cell lines (MSTO-211H, H28, H226, ZL5, ZL34, ZL55, SPC111, SPC212, JL1), immortalized mesothelial cell lines (Met-5A, LP9/TERT-1), colon cancer cell lines (HT-29, Caco-2), embryos from C57Bl/6J mice at embryonic day 10.5, and primary mesothelial cells from wild-type and calretinin-deficient (CR−/−) mice
• Exposure: Butyrate (Bt) applied to cells, generally 0.5–2 mM for 48 or 72 hours (1 mM in reporter assays). Calretinin and septin 7 levels were also changed experimentally by overexpression or shRNA. No asbestos exposure was tested.
• Main results:
  • In all tested cell lines (MSTO-211H, Met-5A, ZL5, ZL55, SPC212, ZL34), butyrate treatment increased calretinin levels in a concentration-dependent way. Calretinin rose by +290% in MSTO-211H cells and +60% in ZL55 cells.
  • In MSTO-211H cells after butyrate, reporter activity rose by 41 ± 16% with the full-length fragment. It reached 200 ± 24% of control with the BRE9–13 fragment, 109 ± 9% with BRE10–13 and 159 ± 14% with BRE9–11. In ZL55 cells the maximum was 123 ± 18% with BRE9–13.
  • In Caco-2 colon cancer cells, the BRE9–13 fragment gave a small increase (+19 ± 16%) that was not statistically significant.
  • In extracts from butyrate-treated MSTO-211H cells, less septin 7 was bound to the BRE9–13 DNA fragment (mass spectroscopy: −34%).
  • Overexpressing septin 7 in MSTO-211H cells lowered calretinin; a GFP control virus had no effect. Lowering septin 7 with shRNA strongly inhibited cell growth. It did not noticeably change calretinin in the surviving cells.
  • Overexpressing calretinin lowered septin 7 levels in MSTO-211H cells, and also in SPC212 mesothelioma cells and HT-29 colon cancer cells.
  • In mouse embryos at embryonic day 10.5, calretinin and septin 7 staining overlapped almost completely in the mesenchyme. Cultured mesothelial cells from CR−/− mice had clearly higher septin 7 than cells from wild-type mice.
  • In MSTO-211H-CR and ZL55-CR cells, an anti-calretinin antibody pulled down septin 7 (co-immunoprecipitation). In dividing cells the two proteins partly colocalized at the cleavage furrow and midbody.
• The authors' conclusion: The authors concluded that septin 7 acts as a butyrate-dependent gene regulatory protein binding to the CALB2 promoter, and that calretinin and septin 7 negatively regulate each other. They wrote that targeting septin 7, directly or as an indirect way of targeting calretinin, might represent another strategy for developing mesothelioma therapy.
• Limitations the authors state:
  • The reverse experiment, lowering calretinin to see the effect on septin 7, was not conclusive because the cells died massively.

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: Calretinin, Mesothelioma, Asbestos Exposure, Mesothelial Cells.

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